Methods of nourishing animals

ABSTRACT

The present invention relates to methods of maintaining, and methods of restoring, a desired calcium homeostasis in a non-human terrestrial animal by administering an effective amount of one or more Calcium-Sensing Receptor modulators (CaSRs) to the animal. The invention further provides methods of reducing foot lesions in chickens. The invention also relates to food compositions useful in the methods of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/993,783, which is the U.S. National Stage Application ofInternational Application No. PCT/US2009/003165 filed on May 22, 2009,published in English, which claims the benefit of U.S. ProvisionalApplication No. 61/128,619, filed on May 22, 2008. The entire teachingsof the above applications are incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

-   a) File name: 22132021004SEQLIST.txt; created Mar. 13, 2014, 64 KB    in size.

BACKGROUND OF THE INVENTION

Animal husbandry, the agricultural practice of breeding and raisinglivestock and other domesticated animals, is a major food-producingindustry world-wide. Poor development and growth, disease and evenmorbidity and mortality of these animals are often caused by poornourishment (e.g., poor nutrition, poor nutrient absorption and/orutilization), particularly when the animals are young. Accordingly,there is a need for better methods of animal husbandry that can bepracticed by various sectors of this industry.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the detection of CalciumSensing Receptor (CaSR) proteins in various tissues of animals, and thefinding that different CaSR modulators can alter the expression of CaSRreceptors in these tissues, thereby influencing the physiology, growthand development of the animal.

The present invention provides, in one embodiment, a method ofmaintaining a desired calcium homeostasis in a non-human terrestrialanimal that is administered an agent that adversely affects calciumhomeostasis in the animal. The method comprises co-administering theagent and one or more CaSR modulator(s) to the animal in an effectiveamount to maintain calcium homeostasis to a desired level in the animal.

The invention also provides, in another embodiment, a method ofrestoring a desired calcium homeostasis in a non-human terrestrialanimal that has been administered an agent that adversely affectscalcium homeostasis in the animal. The method comprises administeringone or more CaSR modulator(s) to the animal in an effective amount torestore calcium homeostasis in the animal.

In yet another embodiment, the invention relates to a method ofpreventing foot (e.g., foot pad) lesions in a terrestrial avian animal.The method comprises administering to an avian animal that has ingested,is ingesting, or will ingest an agent that adversely affects calciumhomeostasis resulting in foot pad lesions, one or more CaSR modulator(s)in an effective amount to prevent foot pad lesions in the animal. In apreferred embodiment, the terrestrial avian animal is a chicken.

The invention further relates to a method of weaning a young pig,comprising administering one or more CaSR agonist(s) to the pig in aneffective amount to agonize one or more Calcium-Sensing Receptors(CaSRs) in the gastrointestinal tract of the pig.

In addition, the invention provides a method of improving the skin of anavian animal, comprising administering one or more CaSR antagonist(s)and a source of Vitamin D in effective amounts to antagonize one or moreCalcium-Sensing Receptors (CaSRs) in the skin of the animal.

The invention also relates to a method of inhibiting an entericcondition in a non-human terrestrial animal, comprising administeringfendiline to the animal in an effective amount to modulate one or moreCalcium-Sensing Receptors (CaSRs) in the gastrointestinal tract of theanimal.

The invention further relates to a food composition for non-human animalconsumption, comprising at least one chelated mineral compound in anamount that adversely affects calcium homeostasis in the animal and atleast one CaSR modulator in an effective amount to maintain and/orrestore a desired calcium homeostasis in the animal.

In addition, the invention relates to a food composition for chickenconsumption, comprising at least one agent in an amount that adverselyaffects calcium homeostasis in the animal, 25-hydroxycholecalciferol ata concentration of about 0.05% by weight and a source of calcium.

The methods and feeds of the present invention can be used to produceanimals with improved health and growth, as well as other beneficialtraits, relative to many methods and feeds that are currently employedin animal husbandry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image of an electrophoretic gel showing a 653 bp fragment(open arrow) containing a CaSR sequence that was amplified from chickintestine by reverse-transcriptase polymerase chain reaction (RT-PCR).Molecular weight standards are shown in the left lane, with thepositions of the 603 bp and 872 bp standards indicated by black arrows.

FIGS. 2A-C show the complete nucleotide sequence (SEQ ID NO:1) of achick jejunum CaSR cDNA transcript.

FIG. 3 shows the predicted amino acid sequence (SEQ ID NO:2) encoded bythe CaSR cDNA nucleotide sequence shown in FIG. 2. All amino acids aredenoted by single letter standard code.

FIG. 4 is a Southern blot of chicken genomic DNA that was digested withEcoRI and probed with ³²P-labeled cDNA containing chick intestinal CaSRcDNA sequence prepared from the sequence shown in FIG. 2. A 5.2 kb EcoRIfragment hybridizes to the CaSR cDNA sequence.

FIGS. 5A and B show the aligned amino acid sequences of calciumreceptors that have been cloned from human parathyroid (SEQ ID NO:3),chicken jejunum (SEQ ID NO:4), salmon kidney (SEQ ID NO:5), cod kidney(SEQ ID NO:6), shark kidney (SEQ ID NO:7) and lobster genomic DNA (SEQID NO:8). Conserved cysteine residues are indicated by vertical boxes.Amino acid residues that are partially conserved (i.e., conserved insome but not in all six of the sequences) are underlined.

FIG. 6 is an immunoblot depicting the detection of CaSR proteins in HEKcell homogenates using a specific anti-CaSR antiserum. Lane 1:homogenate of HEK cells that were stably transfected with a human CaSRcDNA; Lanes 2 and 3: homogenates of HEK cells transfected withlinearized chicken jejunum CaSR from two different transfectionexperiments. The open arrow indicates the position of a broad ˜130 kDimmunoreactive band in lane 1, while the black arrow indicates theposition of two closely-spaced, similarly-sized immunoreactive bands inboth lanes 2 and 3.

FIG. 7A is an image showing immunocytochemical staining of HEK cellsthat were transfected with a full length cDNA of chick intestinal CaSRusing pre-immune serum. Damaged or dead cells that are nonspecificallylabeled are indicated by the open arrows.

FIGS. 7B-D are images showing immunocytochemical staining of HEK cellsthat were transfected with a full length cDNA of chick intestinal CaSRusing anti-CaSR antiserum. Downward pointing arrows indicate thepresence of “nests” of cells that are labeled by anti-CaSR antiserum.Damaged or dead cells that are nonspecifically labeled are indicated bythe open arrows.

FIG. 8 is an immunoblot depicting the detection of phospho-ERK kinase inhomogenates of HEK cells that were exposed to an increase inextracellular calcium using an antibody that is specific for phospho-ERKkinase, but does not recognize the dephosphorylated form of ERK kinase.Lane 1: HEK cells stably transfected with human CaSR cDNA and expressingrecombinant human CaSR protein; Lane 2: HEK cells—non transfectedcontrol; Lane 3: HEK cells transfected with linearized chick jejunumCaSR cDNA and expressing recombinant chicken CaSR protein; Lane 4: HEKcells transfected with linearized chick jejunum CaSR cDNA in a differenttransfection experiment than those in Lane 3 and expressing recombinantchicken CaSR protein. Open arrow indicates phospho-ERK proteins in lane1, while black arrow indicates phospho-ERK proteins in lanes 3 and 4.

FIG. 9A is a Northern blot of mRNA isolated from segments of chickintestine or kidney using a 32P-labeled full-length CaSR cDNA probe fromchick jejunum. Lanes: 1) proximal ⅓ of intestine 2) middle ⅓ ofintestine; 3) distal ⅓ of intestine and 4) kidney.

FIG. 9B is the same blot shown in FIG. 9A after being stripped andreprobed with a beta-actin probe to assess differences in mRNA lanecontent.

FIG. 10 is an image depicting immunolocalization of CaSR protein in asection of proximal intestine from a 10 week old chicken using anti-CaSRantiserum. The left-pointing solid arrows indicate localization of CaSRsin the mucosal layer of the intestine while the open arrows show CaSRprotein in the area of the crypts. The right-pointing solid arrowsindicate the absence of significant CaSR staining in the area of thesubmucosa.

FIG. 11A is an image depicting immunolocalization of CaSR protein in asection of proximal intestine from a newly hatched chicken. The presenceof CaSR protein is indicated by a gray reaction product and arrows.

FIG. 11B is an image depicting immunolocalization of CaSR protein in asection of distal intestine from a newly hatched chicken. The presenceof CaSR protein is indicated by a gray reaction product and arrows.

FIG. 11C is an image depicting immunolocalization of CaSR protein in asection of proximal intestine from a 5 day old chicken. The presence ofCaSR protein is indicated by a gray reaction product and arrows.

FIG. 11D is an image depicting immunolocalization of CaSR protein in asection of proximal intestine from a 10 week old chicken. The presenceof CaSR protein is indicated by a gray reaction product and arrows.

FIG. 12 is an image of an electrophoretic gel showing steady state CaSRmRNA levels, as measured by PCR, in the proximal intestine (Prox. Int.)or distal intestine (Distal Int.) of newly hatched chicks that weregiven water with no added CaSR modulators (con), or water to which theCaSR modulators calcium and tryptophan were added (Trp/Ca) for 1 or 2days.

FIG. 13A is a bar graph depicting plasma calcium levels (Y-axis) in twoweek old male chicks that were gavaged either with a single 3 mg dose ofMC 0100 at various intervals of time after gavage (X-axis). Asterisksindicate a significant difference in mean value as compared to thecontrol.

FIG. 13B is a bar graph depicting plasma calcium levels (Y-axis) in twoweek old male chicks that were gavaged either with a single 3 mg dose ofMC 0100, or water alone, at various intervals of time after oral gavage(X-axis). Asterisks indicate a significant difference in mean value ascompared to the control.

FIG. 14 is a bar graph from a dose response study depicting plasmacalcium levels (Y-axis) in two week old chicks that were gavaged withdifferent doses of MC 0100 or water alone. Asterisks indicate asignificant difference in mean value as compared to water the control.

FIGS. 15A and B are bar graphs from a dose response study depictingplasma calcium levels (Y-axis) in newly hatched chicks at various timesafter oral gavage with a single 1 mg dose of MC 0100 or water alone.Asterisks indicate a significant difference in mean value as compared tothe control.

FIG. 16 is a bar graph from a dose response study depicting plasmacalcium levels (Y-axis) in newly hatched chicks two hours after a 0.25ml oral gavage with different doses of MC 0100 in water or 20%cyclodextrin carrier. Asterisks indicate a significant difference inmean value as compared to water the control.

FIG. 17 is a bar graph from a dose response study depicting plasmacalcium levels (Y-axis) in two week old chicks two hours after a 0.25 mloral gavage with different doses of MC 0100 or water alone. Asterisksindicate a significant difference in mean value as compared to water thecontrol. The highest dose included the use of cyclodextrin carrier thatagain interfered with the action of MC 0100.

FIGS. 18A and B are images of fetal pig gastrointestinal tissue thatshow localization of CaSR protein (see rose-colored reaction product andarrows) to epithelial cells lining mucosal surfaces of the intestine, asdetermined by immunocytochemistry using anti-CaSR antibody.

FIG. 19A is a tissue section of the antrum from a 15-16 day old femalepiglet after 9 hours of weaning, which shows localization of CaSRprotein, as determined by immunocytochemistry using anti-CaSR antibody.

FIG. 19B is a tissue section of the pyloric region of the stomach of a15-16 day old female piglet after 9 hours of weaning, which showslocalization of CaSR protein (see rose-colored reaction product) asdetermined by immunocytochemistry using anti-CaSR antibody. The lumen ofthe stomach is the space at the left of the panel.

FIG. 19C is a tissue section of the duodenum of a 15-16 day old femalepiglet after 9 hours of weaning, which shows localization of CaSRprotein, as determined by immunocytochemistry using anti-CaSR antibody.The lumen of the stomach is the space at the left of the panel.

FIG. 20A is a bar graph depicting the concentration of the polyaminesspermine, spermidine and putrescine in sow's milk at various time pointsduring lactation.

FIG. 20B is a listing of the concentrations of the ionic constituents ofsow's milk and colostrum.

FIG. 20C is a graph depicting how recombinant CaSR protein expressed byHEK cells is activated by concentrations of spermine and calcium thatare present in sow's milk, as shown in FIG. 20B.

FIG. 21A is a graph depicting the effect of casein and bovine serumalbumin (BSA) on activation of recombinant human CaSR protein expressedin HEK cells.

FIG. 21B is a graph depicting the effect of the peptidesglycine-glycine-arginine (gly-gly-arg) and glycine-phenylalanine(gly-phe) on activation of recombinant human CaSR protein expressed inHEK cells.

FIG. 22 is a graph depicting the effect of diaminopropane, a smallpositively charged organic molecule, on activation of recombinant humanCaSR protein expressed in HEK cells.

FIG. 23A shows the chemical structure of spermine(N-[4-[1-(3-Aminopropyl)-2-hydrox-2-nitrosohydrazino]butyl-1,3-propanediamine).

FIG. 23B shows the design of a polyamine (spermine) polymer and productsgenerated by hydrolysis of the polymer.

FIG. 24 is a schematic diagram illustrating how CaSRs that are presenton the mucosal surfaces of the intestine and stomach of young animalscan be modulated by CaSR modulators provided in the diet of a younganimal. The gray-colored reaction product shows the localization ofCaSRs in a section of animal intestine that has been stained withanti-CaSR antiserum. The lumen of the intestine is in the center of thediagram. The mucosal surface of the intestine is intensely labeled byanti-CaSR staining, and the epithelial cells containing the CaSR proteinare constantly exposed to the luminal contents of the intestine. TheCaSRs present in the intestine are modulated by ingested CaSR modulatorspresent in the luminal contents of the gastrointestinal tract. A secondgroup of CaSR modulators modulate CaSRs present in the mucosalepithelial cells.

FIG. 25A depicts the chemical structure of the phenylalkylaminecalcimimetic compound MC 0100, also known as NPS-R-467.

FIG. 25B depicts the chemical structure of the phenylalkylaminecalcimimetic compound MC 106, also known as NPS-2143.

FIGS. 26A-D are graphs showing that the CaSR agonists, MC0100 (acalcimimetic) or 2.5 mM Ca2+, potentiate the response of the mammalianCaSR to the chelated mineral—Mintrex Zn. All tracings shown in FIGS.26A-D were obtained from a single aliquot of CaSR transfected cellsanalyzed on the same day.

FIG. 26A: After an initial addition of 2.5 mM Ca2+ the mammalian CaSRshows a response to the addition of 0.025% Mintrex Zn. Subsequentaddition of Ca2+ to a final concentration of 5 mM produces an additionalresponse.

FIG. 26B: After addition of experimental buffer (EB), no response isobserved upon addition of 0.025% Mintrex Zn.

FIG. 26C: After 2 additions of EB, Ca2+ is added to a finalconcentration of 5 mM.

FIG. 26D: Addition of the CaSR modulator, MC0100 to a finalconcentration of 1 micromolar does produce a very small CaSR responseand subsequent addition of 0.025% Mintrex Zn now elicits a response asdoes a subsequent addition of Ca2+ to a final concentration of 5 mM.

FIG. 27 is a graph showing that the CaSR agonist MC0100 (a calcimimetic)potentiates the response of the avian CaSR to the chelated mineralMintrex Zn. As shown in the Control tracing indicated by the opendiamonds, 2 additions of experimental buffer (EB) to cells suspended inbuffer containing 1.5 mM Ca2+ do not produce a response by the avianCaSR. However, after addition of 7.5 mM Ca2+ to the same cellsuspension, there is a response by the avian CaSR as indicated by thelarge upward deflection of the curve. By contrast, as indicated by theopen squares, after an identical addition of EB, addition of 0.25%Mintrex Zn produces a response from the CaSR followed by a diminishedresponse to the addition of Ca2+ to a final concentration of 7.5 mM. Athird experimental run from the same collection of cells denoted bysolid circles yielded a response upon addition of MC0100 to a finalconcentration of 1 micromolar followed by a significantly largerresponse of the avian CaSR upon addition of 0.25% Mintrex Zn. However,the subsequent CaSR response to Ca2+ addition to 7.5 mM was reduced. All3 tracings are derived from a single pool of cells.

FIGS. 28A and B are graphs showing that the CaSR antagonist, MC106 (acalcilytic) reduces or eliminates the CaSR response to either Ca2+ orthe chelated mineral, Mintrex Zn.

FIG. 28A: The tracing indicated by the open diamonds shows that after aninitial addition of dimethylsulfoxide (DMSO) addition of Ca2+ to a finalconcentration of 2.5 mM elicits a response by the mammalian CaSR that isalso present upon subsequent addition of Ca2+ to a final concentrationof 5 mM. By contrast, as shown by the solid squares, addition of 1micromolar MC 106 suspended in the same concentration of DMSO does notitself produce a CaSR response but eliminates the CaSR response to 2.5mM Ca2+ and reduces the subsequent response to 5 mM Ca2+.

FIG. 28B: The tracing indicated by open circles shows that addition ofDMSO elicits no CaSR response itself while subsequent addition of 0.25%Mintrex Zn and 5.5 mM Ca2+ produce responses by the mammalian CaSR. Bycontrast, as shown by the solid triangles, initial addition of 1micromolar MC 106 in the same concentration of DMSO as before does notelicit a CaSR response but also reduces or eliminates responses by theCaSR to either 0.25% Mintrex ZN or 5.5 mM Ca2+. The final addition ofthe detergent Triton X-100 serves as an internal control in that itlyses the HEK cells and liberates all FURA2 dye for calibrationpurposes.

FIG. 29A is a graph showing that the CaSR modulator MC0100 potentiatesthe response of the avian CaSR to the chelated mineral amino acidcomplex, ZnPro. As shown in the Control tracing (solid diamonds), 2additions of experimental buffer (EB) to cells suspended in buffercontaining 0.5 mM Ca2+ do not produce a response by the avian CaSR. Bycontrast, after addition of the CaSR modulator, MC0100 (solid squares oropen triangles) followed by addition of either 0.02% ZinPro (opentriangles) or 0.2% ZnPro (solid squares) produced responses from avianCaSRs (indicated by upward deflections). Subsequent addition of Ca++ toa final concentration of 5 mM also produced a response. In all aliquots,the detergent Triton X-100 was added as the last addition to lyse thecells.

FIG. 29B is a graph showing that the CaSR modulator MC0100 potentiatesthe response of the mammalian CaSR to a zinc proteinate. Two individualaliquots from a single pool of HEK cells that stably express therecombinant mammalian CaSR protein were used for ratio imagingfluorimetry assays of CaSR activation as described previously. As shownin the tracing indicated by open diamonds, addition of Ca++ to a finalconcentration of 2.5 mM produced a CaSR response (upward deflection)that was also followed by responses after subsequent additions of 0.025%Zn proteinate followed by Ca++ to a final concentration of 7.5 mM. Bycontrast, addition of 1 micromolar MC0100, a CaSR modulator (shown intracing with solid squares) did not itself produce a large response butinstead increased the response of the CaSR to the same dose of 0.025% ZnProteinate. There was also a response to a subsequent dose of Ca++ to afinal concentration of 7.5 mM. In both tracings, the detergent TritonX-100 was added as a final step to lyse the cells.

FIGS. 30A-D are graphs depicting a comparison of the effects of the CaSRmodulators MC0100 and MC106 on avian vs. mammalian CaSRs stimulated byeither zinc proteinate or the zinc organic acid complex, Mintrex Zn.Multiple aliquots of HEK cells stably expressing either the avian CaSR(FIGS. 30A and C) or mammalian CaSR (FIGS. 30B and D) were used toperform ratio imaging fluorimetry to determine the effects of CaSRmodulators MC0100 or MC 106 on CaSR stimulation by chelated minerals.

FIG. 30A: Prior addition of Ca++ to a final concentration of 2.5 mM(solid squares) or 1 micromolar MC0100 (open triangles) produced asimilar response of the avian CaSR as did experimental buffer (EB) alone(solid diamonds) when 0.25% Zinc Proteinate was then added to the cells.Subsequent addition of additional Ca++ to a final concentration of 7.5mM to any of the 3 aliquots (EB alone; MC0100 or 2.5 mM Ca++) producedlittle or no CaSR response. By contrast, prior addition of 1 micromolarMC106 (open circles) reduced the avian CaSR's response to Zn Proteinate.

FIG. 30B: Similar analyses of the effects of prior addition of Ca++,MC0100 or MC 106 on the response of the mammalian CaSR to ZincProteinate. The pattern of inhibition by MC106 on the CaSR response toZinc Proteinate was similar to the pattern displayed in FIG. 30A.

FIG. 30C: Similar analysis of the effect of pre-addition of MC106 on theavian CaSR on stimulation with zinc organic acid chelate, Mintrex Zn.

FIG. 30D: Similar analysis of the effect of MC106 to suppress thestimulation of the mammalian CaSR by the zinc organic acid chelate,Mintrex Zn.

FIGS. 31A-D are graphs showing that the divalent metal ion-proteinatecomplex termed Zn Proteinate, activates both the avian and mammalianCaSRs in a manner similar to that produced by the divalent metal ionorganic acid complex termed Mintrex Zn in a dose response relationship.

FIG. 31A: Three individual aliquots from a single pool of HEK cells thatstably express the recombinant avian CaSR protein were used for ratioimaging fluorimetry assays of CaSR activation as described previously.After a standard addition of Ca++ to a final concentration of 2.5 mM,either 0.0025% (solid squares) or 0.025% (open diamonds) or 0.25% ZincProteinate was added followed by a second addition of Ca++ to a finalconcentration of 7.5 mM. Lastly, an aliquot of the detergent TritonX-100 was added to lyse the cells. 0.0025% Zinc Proteinate elicitslittle or no CaSR response while 0.025% and 0.25% Zinc Proteinateproduces increasing responses from the avian CaSR. The magnitude of thesubsequent CaSR response to Ca++ was reduced by increasing Zn Proteinatestimulation.

FIG. 31B: Identical analyses as performed in FIG. 31A with Zn Proteinateexcept using HEK cells stably expressing the mammalian CaSR. Themammalian CaSR displays a similar dose response pattern as the avianCaSR.

FIG. 31C: Identical analyses as performed in FIGS. 31A and B exceptusing the zinc organic acid chelate, Mintrex Zn, at concentrations of0.025% (open squares) and 0.25% (solid diamonds) using HEK cells stablyexpressing the avian CaSR. A similar dose response relationship ascompared to Zn proteinates and the avian and mammalian CaSRs wasobserved.

FIG. 31D: Identical analyses as performed in FIG. 31C using the zincorganic acid chelate, Mintrex Zn, at concentrations of 0.025% (opensquares) and 0.25% (solid diamonds) on HEK cells stably expressing themammalian CaSR. Mintrex Zn exhibits a dose response relationship withMintrex Zn similar to that displayed by the avian CaSR in FIG. 31C.

FIG. 32 is a graph showing that the addition of 3 mM L-Phenylalanine(L-Phe) reverses Mintrex-Zn's inhibition of the avian CaSR's response toa Ca++ stimulus. As shown by the open squares, prior addition of 0.25%Mintrex Zn to the recombinant chicken CaSR expressed in HEK cellsproduces an initial receptor response (indicated by upward deflection)but then no CaSR response is obtained after the addition of Ca++ to afinal concentration of 7.5 mM. The detergent Triton X-100 was added lastto lyse the cells. By contrast, identical fluorimetry analysis of aseparate aliquot from the same pool of cells as shown by the soliddiamonds, shows that addition of 3 mM L-Phe after CaSR stimulation with0.25% Mintrex Zn now produces a response to addition of Ca++ to 7.5 mM.Thus, prior addition of L-Phe reverses or rescues the avian CaSR fromits inhibition by the chelated mineral Mintrex Zn.

FIG. 33 is a graph showing that the addition of 3 mM L-Phenylalanine(L-Phe) reverses Mintrex-Zn's inhibition of the mammalian CaSR'sresponse to a Ca++stimulus at extracellular calcium concentrations thatcorrespond to mammalian serum (1.5 mM Ca++). As shown by the opensquares, addition of experimental buffer alone (EB) produces no responseby the recombinant mammalian CaSR expressed in HEK cells. However,addition of 0.025% Mintrex Zn produces an initial receptor response(indicated by upward deflection) but then no CaSR response is obtainedafter the addition of Ca++ to a final concentration of 5 mM. Thedetergent Triton X-100 was added last to lyse the cells. By contrast,identical fluorimetry analysis of a separate aliquot from the same poolof cells as shown by the solid diamonds, shows that addition of 3 mML-Phe after CaSR stimulation with 0.025% Mintrex Zn now produces aresponse to addition of Ca++ to 5 mM. Thus, prior addition of L-Phereverses or rescues the mammalian CaSR from its inhibition by thechelated mineral Mintrex Zn in a manner similar to that shown for theavian receptor in FIG. 32.

FIGS. 34A and B are Western blots showing that the avian CaSR protein isable to respond to metal chelates via activation of downstream signalingpathways like CaSR dependent ERK 1/2 phosphorylation. Arrows denote thebands corresponding to phosphorylated ERK1/2 protein. As shown, the ZnProteinate and Mn Proteinate resulted in an increase in ERK1/2phosphorylation with increasing concentration (FIG. 34A, left and rightpanels). By contrast, Cu Proteinate displayed the highest ERK1/2phosphorylation after exposure of HEK cells to the lowest concentrationof 0.025% (FIG. 34B). The calcimimetic MC 0100 strongly enhanced theERK1/2 phosphorylation response to 0.025% Mn Proteinate and to a lesserdegree to Zn Proteinate. Note that Zn Proteinate at 0.025% alreadyshowed a higher phosphorylation response when compared to the responseto 0.025% Mn Proteinate. By contrast, the addition of MC 0100 incombination with 0.025% Cu Proteinate results in a net reduction of theERK1/2 phosphorylation response when compared to other metal chelatecomplexes tested.

FIG. 35 is a Western blot showing that the avian CaSR protein is able torespond to the growth promoting antibiotic Bacitracin Zn via theactivation of downstream signalling pathways like CaSR dependent ERK 1/2phosphorylation. Arrows denote the bands corresponding to phosphorylatedERK1/2 protein. As shown, exposure of HEK cells stably expressing thechicken CaSR to bacitracin Zn resulted in an increase in ERK1/2phosphorylation as compared to Ca++ with MC0100.

FIG. 36 is a graph showing that fendiline stimulates the avian CaSRsimilar to the responses observed for the CaSR agonist MC0100.Fluorimetry analysis was performed on HEK cells expressing therecombinant chicken CaSR protein. As shown by the open trianglessymbols, after addition of 2.5 mM Ca++, a series of stepwise additionsof the known CaSR modulator, MC0100, causes increased responses of theCaSR as indicated by the series of upward deflections and elevation ofthe baseline. In a similar manner, additions of fendiline (shown by thesolid squares) to a second aliquot of the same CaSR-HEK cells producesimilar responses in the avian CaSR. As a control, no such responseswere observed when additions of the vehicle DMSO only was added asindicated by the solid diamonds followed by a single addition of Ca++ toa final concentration of 5 mM. The addition of the detergent TritonX-100 was added last to each cuvette to lyse the cells at the end ofeach analysis run.

FIG. 37 is a graph showing that fendiline stimulates the mammalian CaSRsimilar to the responses observed for the CaSR agonist MC0100.Fluorimetry analysis was performed on HEK cells expressing therecombinant mammalian CaSR protein. As shown by the open diamondsymbols, after addition of 2.5 mM Ca++, a series of stepwise additionsof the known CaSR modulator, MC0100, causes increased responses of theCaSR as indicated by the series of upward deflections and elevation ofthe baseline. In a similar manner, additions of fendiline (shown by thesolid squares) to a second aliquot of the same CaSR-HEK cells producesimilar responses in the avian CaSR. As a control, no such responseswere observed when additions of the vehicle DMSO only was added asindicated by the solid diamonds followed by a single addition of Ca++ toa final concentration of 5 mM. The addition of the detergent TritonX-100 was added last to each cuvette to lyse the cells at the end ofeach analysis run.

FIG. 38 is a graph showing that prenylamine stimulates the avian CaSRsimilar to the responses observed for other CaSR agonists. Fluorimetryanalysis was performed on HEK cells expressing the recombinant chickenCaSR protein. Analysis was performed on 3 separate aliquots obtainedfrom a single pool of CaSR-HEK cells using either 1 micromolar (solidtriangles), 3 micromolar (open circles) or 5 micromolar (solid squares)prenylamine after addition of Ca++ to a final concentration of 2.5 mM.After the addition of prenylamine, each aliquot of cells received asubsequent addition of Ca++ to a final concentration of 7.5 mM. A CaSRresponse is shown as an upward deflection and elevation of the baseline.As a control, no such response was observed when addition of the vehicleDSMO (shown as the open diamonds) was added. The addition of thedetergent Triton X-100 was added last to each cuvette to lyse the cellsat the end of each analysis run.

FIG. 39 is a graph showing that prenylamine stimulates the mammalianCaSR similar to the responses observed for other CaSR agonists.Fluorimetry analysis was performed on HEK cells expressing therecombinant mammalian CaSR protein. Analysis was performed on 3 separatealiquots obtained from a single pool of CaSR-HEK cells using either 1micromolar (solid diamonds), 3 micromolar (open circles) or 5 micromolar(solid squares) prenylamine after addition of Ca++ to a finalconcentration of 2.5 mM. After the addition of prenylamine, each aliquotof cells received a subsequent addition of Ca++ to a final concentrationof 7.5 mM. A CaSR response is shown as a upward deflection and elevationof the baseline. As a control, no such response were observed whenaddition of the vehicle DSMO (shown as the open circles) was added. Theaddition of the detergent Triton X-100 was added last to each cuvette tolyse the cells at the end of each analysis run.

FIGS. 40A-C are images of fixed tissue sections of broiler chicken skinfrom the foot pad region of broiler chicken feet depicting thelocalization of calcium sensing receptor protein (CaSR) in epidermal anddermal tissues. Standard immunocytochemistry was performed onpermeabilized fixed tissue sections of broiler chicken skin from thefoot pad region of feet using either specific anti-CaSR antiserum (FIGS.40A and C) or pre-immune anti-CaSR antiserum (FIG. 40B). The presence ofbound anti-CaSR antibody (dark areas of staining) that denotes thelocation of CaSR protein is indicated by the arrows shown in FIG. 40A(see staining present in stratum corneum, epidermis and dermal skinregions) and FIG. 40C (see areas of intensely stained cells withindermis). FIG. 40B: No dark areas of staining were observed when usingthe pre-immune antiserum.

FIG. 41 is a graph depicting a comparison of the effects of inclusion ofthe CaSR modulators MC0100 and MC106, or Mintrex Zn, on the serumcalcium in broiler chickens under nonstressed pen trial conditions. Ascompared to Control values, significant reductions in the mean serumcalcium concentration were observed for the two groups of birdsreceiving either MC0100 or Mintrex Zn as a feed additive. No differencesin serum pH, Na+, K+ or Cl− were observed for any of the groups tested.

FIG. 42A is a schematic showing the division of a total of 1080 maleCobb strain birds into 5 different treatment groups that were subjectedto identical rearing conditions that included being reared using dirtylitter conditions to simulate standard large scale chicken rearingfacilities.

FIG. 42B is a graph showing a comparison of performance parameters forbroiler chicken groups shown in FIG. 42A, including mean values for bothaverage weight and adjusted feed conversion ratio (FCR) in the animalsfrom 5 treatment groups. Control Group A received standard feeds whichincluded 100 ppm inorganic zinc throughout the 42 day grow out. GroupsB-E were subjected to a 10 day interval of zinc depletion followed byzinc repletion using different sources of dietary zinc (Group B—40 ppminorganic zinc), Mintrex Zn (Group C—20 ppm Zinc), Mintrex Zn +MC0100(Group D—20 ppm Zinc; 70 ppm MC0100) or Mintrex Zn+MC106 (Group E—20 ppmzinc; 70 ppm MC 106). While there were no significant differences in themeans between the 5 groups, Group E achieved the largest mean bodyweight and lowest FCR.

FIG. 43A is a graph showing the frequency of foot pad lesions in broilerchickens for the 5 treatment groups shown in FIG. 42B after 24/25 daysof rearing under dirty litter conditions. The group receiving thecombination of Mintrex Zn+MC 106 displayed a significant reduction inthe severity and frequency of foot pad lesions as compared to all othergroups.

FIG. 43B is a graph showing the severity of foot pad lesions in broilerchickens for the 5 treatment groups shown in FIG. 42B after 24/25 daysof rearing under dirty litter conditions. The severity of the foot padlesions present was graded on a scale from 0 (no lesions present), 1(mild lesions present), 2 (moderate lesions present) and 3 (severelesions present). Mean scores for foot pad severity for each of the 5test groups are shown. There was a significant reduction in the meanfoot pad severity score of the group receiving Mintrex Zn+MC 106 ascompared to all other groups.

FIG. 44A is a graph showing the frequency of foot pad lesions in broilerchickens for the 5 treatment groups shown in FIG. 42B after 42/43 daysof rearing under dirty litter conditions. The 5 groups included aControl group (ZnS) that received standard feeds which included 100 ppminorganic zinc throughout the 42 day grow out. The group receiving thecombination of Mintrex Zn+MC106 displayed a significant reduction in thefrequency of foot pad lesions as compared to all other groups.

FIG. 44B is a graph showing the severity of foot pad lesions in broilerchickens for the 5 treatment groups shown in FIG. 42B after 42/43 daysof rearing under dirty litter conditions. The 5 groups included aControl group (ZnS) that received standard feeds which included 100 ppminorganic zinc throughout the 42 day grow out. The severity of the footpad lesions present was graded on a scale from 0 (no lesions present), 1(mild lesions present), 2 (moderate lesions present) and 3 (severelesions present). Mean scores for foot pad severity for each of the 5test groups are shown. There was a significant reduction in the meanfoot pad severity score of the group receiving Mintrex Zn+MC 106 ascompared to all other groups.

FIG. 45A is a graph showing the ionized calcium of broiler chickens onDay 24/25 of a 42 Day Grow Out study. A total of 1080 Cobb broilerchickens were divided into 5 test groups and reared under conditionsdescribed in FIGS. 42A and B. Mean values for ionized calciumconcentrations for the 5 test groups are shown. The birds receiving thecombination of Mintrex Zn+MC0100 displayed an ionized calciumconcentration that was significantly lower than all other test groups.

FIG. 45B is a graph showing the total serum calcium of broiler chickenson Day 24/25 of a 42 Day Grow Out study. A total of 1080 Cobb broilerchickens were divided into 5 test groups and reared under conditionsdescribed in FIGS. 42A and B. These values correspond to the samesamples used for determination of ionized calcium shown in FIG. 45A. Thebirds receiving the combination of Mintrex Zn+MC106 displayed thehighest total calcium concentrations.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “non-human terrestrial animal” refers to a non-human animalthat lives predominantly or entirely on land. A “non-human terrestrialanimal” can be, for example, a mammal (e.g., a bovine species, a porcinespecies, an equine species) or an avian animal. The animal can be of anyage or at any stage of development.

A “young animal” refers to an animal in a stage of development betweenthe time it is separated from its fetal environment and the time whenrapid growth ceases. Thus, a “young animal” can be, for example, aneonatal, juvenile or adolescent animal. In the case of mammals, a younganimal can be a nursing animal or a weanling. In the case of egg-layingspecies, the young animal can be an animal developing within anunhatched egg or a hatched animal.

The term “Calcium-Sensing Receptor” or “CaSR” refers to any multimodal Gprotein coupled receptor (GPCR) that senses extracellular levels ofcalcium ions and/or other ions (e.g., Zn2+, Mn2+, Cu2+). CaSRs are alsoknown in the art as “polyvalent cation-sensing receptors,” “polyvalentcation receptors” and “PVCRs,” and these terms are used interchangeablyherein. CaSR proteins from different animal species are known in theart. Other CaSR proteins include those identified herein (e.g., SEQ IDNO:3).

“CaSR modulator” refers to any naturally-occurring, recombinant,synthetic or semi-synthetic agent that binds to or otherwise modulatesthe expression, sensitivity, activity, signalling and/or physiologicalfunction of a CaSR protein in one or more tissues of a young animal. A“CaSR modulator” can be, for example, a CaSR agonist, also referred toherein as a calcimimetic, or a CaSR antagonist, also referred to hereinas a calcilytic. The term “CaSR modulator” encompasses primary receptorligands as well as allosteric modulators of a CaSR protein. PreferredCaSR modulators include, for example, polyvalent cations (e.g., divalentcations, trivalent cations, organic polycations), L-amino acids (e.g.,L-aromatic amino acids, L-kynurenines), peptides, phenylalkylamines,polyaromatic hydrocarbons, substituted piperidines and substitutedpyrrolidines. Particularly preferred CaSR modulators include, forexample, peptides (e.g., gly-gly-arg tripeptide, glutathione), divalentcations (Ca²⁺ ions, Mg²⁺ ions), aromatic amino acids (e.g., trytophan),polyamines (e.g., spermine, spermidine, putrescine, diaminopropane),phenylalkylamines (e.g., MC 0100, fendiline, prenylamine), mineralchelates (e.g., Mintrex Zn) and casein.

“CaSR agonists” include Type I agonists/calcimimetics, which do notrequire Ca2+ to agonize a CaSR, and Type II agonists/calcimimetics,which require Ca2+ to agonize a receptor.

As used herein, the “sensitivity” of the CaSR refers to alteration ofCaSR expression in response to a change in the concentration of CaSRmodulators or an alteration in the ability of the CaSR to respond tovarious ligands that stimulate its interaction with other cellularsignal transduction pathways. CaSR expression can be assessed bymeasuring or detecting CaSR polypeptide or nucleic acid molecules in asample by standard methods.

An “effective amount” as defined herein refers to an amount orconcentration of one or more CaSR modulator(s) sufficient to achieve adesired effect on an animal that is administered the one or more CaSRmodulator(s) under the conditions of administration. The desired effectcan be, for example, maintenance of calcium homeostasis, restoration ofcalcium homeostasis, enhanced nutrient absorption, and/or utilization,improved growth, inhibition of an enteric condition, reduction in footlesions, promotion of weaning, and improved skin. Preferably, an“effective amount” of a CaSR modulator does not produce significantdetrimental effects associated with excessive CaSR modulation in ananimal, including, but not limited to, toxicity, hypercalcemia,hypocalcemia, reduced appetite, disturbances in electrolyte levels,decreased nutrient utilization and/or fluid retention, and tissuedysfunction (e.g., gastrointestinal tissue dysfunction, nervous tissuedysfunction, endocrine tissue dysfunction).

As used herein, the terms “ionic balance homeostasis” and “mineralbalance homeostasis” are used interchangeably and refer to the mechanismby which essential ions (e.g., Zn, Ca, Cu, Mn) are maintained atadequate levels in an animal's tissues and fluids.

As used herein, the term “calcium homeostasis” refers to the mechanismby which calcium is maintained at adequate levels in an animal's tissuesand fluids. Multiple parameters contribute to calcium homeostasis,including, for example, the concentrations of ions (calcium, magnesium,sodium), amino acids and CaSR modulators in bodily fluids, as well asfluids that bathe various external tissues that express CaSRs, such asthe lining of the gastrointestinal tract. In addition, collections ofstored calcium present in various tissues, such as bone and skin, alsocontribute to calcium homeostasis. Absorptive and excretion mechanismspresent in specific tissues, including the kidney and gastrointestinaltract, further contribute to calcium homeostasis via regulation of theflux of calcium and other CaSR modulators into and out of the body.Calcium sensing receptors (CaSRs) are the principal calciostat affectingcalcium homeostasis in an animal.

As used herein, the expression “maintaining calcium homeostasis” in ananimal means preserving a desired calcium homeostasis in an animal.

As used herein, the expression “desired calcium homeostasis” refers to aphysiological state in which calcium is present in an animal's tissuesand fluids at levels that are sufficient to promote growth, developmentand/or maintenance of skeletal, muscle and fluid constituents in theanimal, or to achieve other desired conditions, characteristics orproperties in the animal.

As used herein, the expression “restoring calcium homeostasis” in ananimal means returning calcium homeostasis to a desired level in ananimal that has been administered one or more CaSR modulators thatadversely affect calcium homeostasis in the animal.

A “CaSR modulator that adversely affects calcium homeostasis” refers toan agent that, when administered to an animal, alters calciumhomeostasis in the animal in a manner that is not beneficial to theanimal's growth, development and/or maintenance of skeletal, muscle orfluid constituents. Such agents can affect a single tissue component(e.g., a CaSR), or several tissue components, that are required tomaintain a desired calcium homeostasis in the animal.

As used herein, the phrase “enhancing nutrient utilization” in an animalmeans increasing the level of nutrients that are absorbed and/orutilized by one or more tissues (e.g., gastrointestinal tissues) of ananimal that receives a diet that is supplemented with additional amountsof a CaSR modulator(s) relative to a suitable control level (e.g., thelevel of nutrients that are absorbed and/or utilized by one or moretissues of an animal that receives a diet that is not supplemented withadditional amounts of a CaSR modulator(s)). The level of nutrients thatare absorbed and/or utilized by one or more tissues in an animal can bedetermined, for example, by determining the difference between the levelof a nutrient in a food source that is ingested by the animal, and thelevel of that nutrient that is excreted in the urine and stool of theanimal. “Enhancing nutrient utilization” includes inhibiting (e.g.,preventing, reducing, treating) one or more enteric conditions thatcontribute to poor nutrient absorption and/or utilization in an animal(e.g., diarrhea, abnormal gut development, impaired nutrientutilization). Nutrient utilization can be assessed, for example, bydetermining the Feed Conversion Ratio (FCR) of the animals. The feedconversion ratio or FCR is obtained by dividing the body weight gainedby a group of animals into the amount of food fed to these animals. Themore efficient the conversion of food into body weight growth byanimals, the smaller the FCR (small amount of food/large weight gain ofanimals). A very small FCR number (less than 1) encompasses a highlyefficient conversion of food into body weight growth and is what theindustry is striving for. By contrast, a large FCR means an inefficientconversion of food into body weight growth and is generally undesirable.A large or poor FCR is undesirable because feed usually is expensive andmore must be used to grow animals to a desired weight.

As used herein, the terms “co-administer,” “co-administered,” and“co-administering” refer to the act of administering a combination oftwo or more agents (e.g., two or more CaSR modulators, an agent thatadversely affects calcium homeostasis in an animal and one or more CaSRmodulators) to an animal either simultaneously or sequentially during ashort period of time (e.g., less than an hour). Two or more agents canbe co-administered, for example, by including the two or more agents inthe same feed that is provided to the animal.

The term “food source” refers to a composition that can be delivered tothe gastrointestinal tract of a young animal and includes nutrientsutilized by the young animal for nourishment. Thus, a “food source” canbe, for example, a feed composition (e.g., a solid feed, a semi-solidfeed), a liquid composition (e.g., an aqueous feed, a non-aqueous liquidfeed, milk), and a nutritional supplement (e.g., a capsule containingvitamins and/or other nutrients).

The term “peptide” refers to a naturally-occurring, synthetic orsemi-synthetic compound that includes from about 2 to about 100 aminoacid residues that are joined together by covalent bonds (e.g., peptidebonds, non-peptide bonds). Such peptides are typically less than about100 amino acid residues in length and are preferably about 2 to about 10amino acid residues in length. Peptides can be linear or cyclic and caninclude unmodified and/or modified amino acid residues. In a preferredembodiment, the peptide comprises amino acids that are joined by peptidebonds. The term “peptide” also encompasses peptidomimetics.

As used herein, the term “polypeptide” refers to a polymer of aminoacids of any length and encompasses proteins, peptides, andoligopeptides.

“Active polymer” refers to a natural, synthetic or semi-syntheticpolymer in a coating that comprises a CaSR modulator as an active agentand releases the active agent under the desired conditions (e.g., theconditions present in a target organ, for example, the stomach or theintestines).

“Weanling” refers to a mammal that is being weaned, or has recently beenweaned (e.g., within a few days or weeks), from it's mother's milk to asolid, semi-solid or liquid diet.

Unless otherwise noted, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art of biology or chemistry (e.g., in cell culture, moleculargenetics, nucleic acid chemistry, hybridization techniques andbiochemistry). Unless otherwise noted, standard techniques are used formolecular, genetic and biochemical methods (see generally, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons,Inc., the relevant teachings of which are incorporated herein byreference) and chemical methods.

Methods of Nourishing Animals; Methods of Modulating Ionic BalanceHomeostasis in Animals

The present invention relates to methods of nourishing animals. Themethods involve modulating the expression, sensitivity, activity,signalling and/or physiological function of a Calcium-Sensing Receptor(CaSR) (e.g., at least one CaSR) in one or more tissues of the animals.In a particular embodiment, the invention relates to modulating theCaSR(s) in gastrointestinal tissues of animals, which provides increasedgrowth and development of the gastrointestinal tract. In anotherpreferred embodiment, the animal is a young animal.

The invention also provides methods of modulating ionic balance, ormineral balance, homeostasis (e.g., calcium homeostasis) in an animal(e.g., a non-human terrestrial animal). In one embodiment, the inventionprovides a method of maintaining a desired calcium homeostasis in anon-human terrestrial animal that is administered an agent thatadversely affects calcium homeostasis in the animal. The methodcomprises co-administering the agent and one or more CaSR modulator(s)to the animal in an effective amount to maintain calcium homeostasis inthe animal. In another embodiment, the invention relates to a method ofrestoring a desired calcium homeostasis in a non-human terrestrialanimal that has been administered an agent that adversely affectscalcium homeostasis in the animal. The method comprises administeringone or more CaSR modulator(s) to the animal in an effective amount torestore calcium homeostasis in the animal.

The methods of the present invention include administering one or moreCaSR modulator(s) to an animal (e.g., adding one or more CaSRmodulator(s) to a food source for an animal (e.g., a young animal), andproviding the animal with the food source). The one or more CaSRmodulator(s) are administered in an effective amount to modulate atleast one CaSR in a tissue of an animal that has ingested the foodsource.

The methods of the present invention can be practiced on animals of anyage, including young, adult and elderly animals. For example, themethods of the invention can be practiced on adult animals, including,but not limited to, adult animals having specialized nutritional needs(e.g., laying hens, lactating cows, pregnant animals). In a preferredembodiment, the animals are young animals.

Particularly suitable animals for the methods of the invention include,but are not limited to, terrestrial animals, including terrestrialmammals and terrestrial avian animals. Preferred terrestrial animalsinclude mammals typically raised as livestock (e.g., cattle, steer,pigs, horses, sheep, deer), as well as companion animals and pets (e.g.,dogs, cats, rabbits, ferrets, hamsters, gerbils, guinea pigs, hedgehogs,birds, lizards, snakes, frogs, toads, turtles, spiders, llamas).Particularly preferred terrestrial animals include cattle (e.g., speciesbelonging to the genus Bos) and pigs (e.g., Sus scrofa domesticus).

Suitable terrestrial avian animals include, for example, chickens,turkeys, ducks, geese, pheasants, grouse, and ostriches. Particularlypreferred terrestrial animals include chickens (e.g., Gallus gallus, G.gallus domesticus) and turkeys.

The animal can be a monogastric animal (e.g., a chicken, a pig) or amultigastric animal (e.g., a cow). The animals can be raised understandard rearing conditions that are known in the art.

CaSRs, which are located in various tissues (e.g., gastrointestinaltissues, for instance, stomach and intestinal tissue) of the animals,sense alterations in levels of CaSR modulators, including variouspolyvalent ions (e.g., divalent cations), for example, in the luminalcontents of the stomach or intestines. The ability to sense CaSRmodulators results in a modulation of the CaSR, thereby allowing theintestines of the animals, and the animals themselves, to grow better,for example by inducing the animals to remodel themselves based on thenutrient and ionic environment that is present in the lumen of thegastrointestinal tract which, in turn, is governed by the type,composition and quality of the food that the young animals consume. Theabilities of CaSRs to sense both specific nutrients (e.g., amino acids,polyamines), as well as specific ions (e.g., Ca2+, Mg2+, Zn2+, Na+)permits controlled development of the gastrointestinal tract and allowsthe animals to grow better. Modulation of the CaSR can occur, forexample, in one or more tissues (e.g., gastrointestinal tissues) of ananimal.

The modulation of CaSRs by CaSR modulators allows for, or assists in,one or more functions in the animals, including but not limited to,sensing or adapting to at least one CaSR modulator in tissues (e.g.,gastrointestinal tissues) or in the surrounding environment; alteringthe behavioral response to sensory stimuli, especially olfaction andgustation; maintaining or altering (e.g., restoring) osmoregulation ordivalent cation (e.g., calcium) homeostasis; altering one or moreendocrine pathways; and altering chemosensory signal concentration orcomposition. CaSR expression can be assessed by measuring or detectingCaSR polypeptide or nucleic acid molecules in a sample by standardmethods. Suitable assays and techniques for assessing the expressionsensitivity, activity, signaling and/or physiological function of a CaSRare known in the art, and include those described herein.

CaSR modulators include both CaSR agonists (e.g., calcimimetics) thatagonize, or increase, the expression, sensitivity, activity, signallingand/or physiological function of at least one CaSR, and CaSR antagonists(e.g., calcilytics) that antagonize, or decrease, the expression,sensitivity, activity, signalling and/or physiological function of atleast one CaSR. Calcimimetic CaSR modulators include, for example, Type1 calcimimetics and Type II calcimimetics (e.g., NPS-R-467 and NPS-R-568from NPS Pharmaceutical Inc., (Salt Lake, Utah, U.S. Pat. Nos.5,962,314; 5,763,569; 5,858,684; 5,981,599; 6,001,884). See Nemeth, E.F. et al., PNAS 95: 4040-4045 (1998)).

CaSR modulators encompass primary receptor ligands for a CaSR, as wellas allosteric modulators of a CaSR (e.g., aromatic amino acids,tryptophan derivatives, peptides). CaSR modulators can be naturallyoccurring (e.g., isolated from a natural source), synthetic (e.g.,produced by standard chemical synthesis techniques), semi-synthetic orrecombinant (e.g., produced by biofermentation).

Suitable CaSR modulators for use in the methods of the inventioninclude, but are not limited to, polyvalent cations (e.g., inorganicpolycations, organic polycations), amino acids, peptides and smallorganic molecules. Examples of inorganic polycations are divalentcations including calcium and magnesium; and trivalent cationsincluding, but not limited to, gadolinium (Gd3+). Typically, monovalentand divalent cations, as well as amino acids, are effective CaSRmodulators when present in the millimolar concentration range, whereastrivalent cations, peptides and small organic molecules typically areeffective CaSR modulators when present in the micromolar concentrationrange.

Examples of organic polycations include, but are not limited to,aminoglycosides such as neomycin or gentamicin, and polyamines (e.g.,polyarginine, polylysine, polyhistidine, polyornithine, spermine,spermidine, cadaverine, putricine, copolymers of polyarginine/histidine, poly lysine/arginine, diaminopropane. See Brown, E.M. et al., Endocrinology 128: 3047-3054 (1991); Quinn, S. J. et al., Am.J. Physiol. 273: C1315-1323 (1997). In a particular embodiment, theorganic polycation is a polyamine.

Additionally, CaSR modulators include amino acids, such as L-aminoacids. The L-amino acids can be unmodified or modified (e.g.,halogenated). Examples of suitable L-amino acids are L-Tryptophan,L-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine, L-Arginine,L-Histidine, L-Leucine, L-Isoleucine, and L-Cystine. See Conigrave, A.D., et al., PNAS 97: 4814-4819 (2000). In a particular embodiment, theL-amino acid is an aromatic amino acid. In a preferred embodiment, theL-amino acid is L-tryptophan. CaSR modulators further includetryptophan-pathway metabolites and tryptophan derivatives, such as, forexample, kynurenine, 3-OH kynurenine, xanthurenic acid, quinolic acidand kynurenic acid.

In addition, suitable CaSR modulators for use in the present inventioninclude peptides. Such peptides are typically less than about 100 aminoacid residues in length, and are preferably about 2 to about 10 aminoacid residues in length (e.g., dipeptides, tirpeptides). The peptide cancomprise any suitable L- and/or D-amino acid, for example, commonα-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g.,β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine,statine), and unusual amino acids (e.g., citrulline, homocitruline,homoserine, norleucine, norvaline, ornithine, kynurenine). The amino,carboxyl and/or other functional groups on a peptide can be free (e.g.,unmodified) or protected with a suitable protecting group. Suitableprotecting groups for amino and carboxyl groups, and methods for addingor removing protecting groups are known in the art and are disclosed in,for example, Green and Wuts, “Protecting Groups in Organic Synthesis”,John Wiley and Sons, 1991. The functional groups of a peptide can alsobe derivatized (e.g., alkylated) using art-known methods.

The peptide can comprise one or more modifications (e.g., amino acidlinkers, acylation, acetylation, amidation, methylation, halogenation,terminal modifiers (e.g., cyclizing modifications)), if desired. Thepeptide can also contain chemical modifications (e.g., N-methyl-α-aminogroup substitution). In addition, the peptide can be an analog of aknown and/or naturally-occurring peptide, for example, a peptide analoghaving conservative amino acid residue substitution(s). Thesemodifications can improve various properties of the peptide (e.g.,solubility, binding), including its ability to modulate a CaSR in ayoung animal.

Peptide CaSR modulators can be linear, branched or cyclic, e.g., apeptide having a heteroatom ring structure that includes several amidebonds. In a particular embodiment, the peptide is a cyclic peptide. Suchpeptides can be produced by one of skill in the art using standardtechniques. For example, a peptide can be derived or removed from anative protein by enzymatic or chemical cleavage, or can be synthesizedby suitable methods, for example, solid phase peptide synthesis (e.g.,Merrifield-type synthesis) (see, e.g., Bodanszky et al. “PeptideSynthesis,” John Wiley & Sons, Second Edition, 1976). Peptides can alsobe produced, for example, using recombinant DNA methodologies or othersuitable methods (see, e.g., Sambrook J. and Russell D. W., MolecularCloning: A Laboratory Manual, 3^(rd) Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001).

Peptides can be synthesized and assembled into libraries comprising afew to many discrete molecular species. Such libraries can be preparedusing methods of combinatorial chemistry, and can be screened using anysuitable method to determine if the library comprises peptides with adesired biological activity. Such peptides can then be isolated usingsuitable methods.

Peptide CaSR modulators can also be peptidomimetic compounds. Forexample, polysaccharides can be prepared that have the same functionalgroups as peptides. Peptidomimetics can be designed, for example, byestablishing the three dimensional structure of a peptide agent in theenvironment in which it is bound or will bind to a target molecule. Thepeptidomimetic comprises at least two components, the binding moiety ormoieties and the backbone or supporting structure.

The binding moieties are the chemical atoms or groups that will react orform a complex (e.g., through hydrophobic or ionic interactions) with atarget molecule, for example, a young animal CaSR. For example, thebinding moieties in a peptidomimetic can be the same as those in apeptide or protein antagonist. The binding moieties can be an atom orchemical group that reacts with the receptor in the same or similarmanner as the binding moiety in the peptide antagonist. For example,computational chemistry can be used to design peptide mimetics of CaSRbinding site, for instance, a ligand binding site. Examples of bindingmoieties suitable for use in designing a peptidomimetic for a basicamino acid in a peptide include nitrogen-containing groups, such asamines, quarternary ammonia moieties, guanidines and amides orphosphoniums. Examples of binding moieties suitable for use in designinga peptidomimetic for an acidic amino acid include, for example,carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, a loweralkyl sulfonic acid ester or a phosphorous acid or ester thereof.

The supporting structure is the chemical entity that, when bound to thebinding moiety or moieties, provides the three dimensional configurationof the peptidomimetic. The supporting structure can be organic orinorganic. Examples of organic supporting structures includepolysaccharides, polymers or oligomers of organic synthetic polymers(such as, polyvinyl alcohol or polylactide). It is preferred that thesupporting structure possesses substantially the same size anddimensions as the peptide backbone or supporting structure. This can bedetermined by calculating or measuring the size of the atoms and bondsof the peptide and peptidomimetic. In one embodiment, the nitrogen ofthe peptide bond can be substituted with oxygen or sulfur, for example,forming a polyester backbone. In another embodiment, the carbonyl can besubstituted with a sulfonyl group or sulfinyl group, thereby forming apolyamide (e.g., a polysulfonamide). Reverse amides of the peptide canbe made (e.g., substituting one or more-CONH-groups for a-NHCO-group).In yet another embodiment, the peptide backbone can be substituted witha polysilane backbone.

These compounds can be manufactured by known methods. For example, apolyester peptidomimetic can be prepared by substituting a hydroxylgroup for the corresponding α-amino group on amino acids, therebypreparing a hydroxyacid and sequentially esterifying the hydroxyacids,optionally blocking the basic and acidic side chains to minimize sidereactions. Determining an appropriate chemical synthesis route cangenerally be readily identified upon determining the chemical structure.

Peptidomimetics can be synthesized and assembled into librariescomprising a few to many discrete molecular species. Such libraries canbe prepared using well-known methods of combinatorial chemistry, and canbe screened to determine if the library comprises one or morepeptidomimetics which have the desired activity. Such peptidomimeticantagonists can then be isolated by suitable methods.

In addition, CaSR modulators include phenylalkylamines. Methods ofsynthesizing, isolating and/or preparing phenylalkylamines are known inthe art. Suitable phenylalkylamines for use in the methods of theinvention include, but are not limited to, the CaSR agonists, MC 0100(See FIG. 25), fendiline, prenylamine and cinacalcet, and the CaSRantagonist MC106. Typically, phenylalkylamine modulators of CaSRactivities are potent when present in the high nanomolar to lowmicomolar range.

CaSR modulators can also be substituted piperidines and substitutedpyrrolidines. Methods of synthesizing, isolating and/or preparingsubstituted piperidines and substituted pyrrolidines are known in theart. Suitable substituted piperidines and substituted pyrrolidines foruse in the methods of the invention include, but are not limited to,substituted piperidines and substituted pyrrolidines described in U.S.Pat. Nos. 7,265,145 and 7,307,171.

CaSR modulators also include compounds that indirectly alter CaSRexpression (e.g., 1,25 dihydroxyvitamin D (e.g., in concentrations ofabout 3,000-10,000 International Units/kg feed), cytokines such asInterleukin Beta, and Macrophage Chemotactic Peptide-1 (MCP-1)).

In addition, CaSR modulators can be chelated mineral compounds.Typically, chelated mineral compounds include a metal cation componentand an organic anion component that serves as a chelator for the metalcation. Preferably, the metal cation component is a divalent metalcation (e.g., Zn2+, Ca2+, Mg2+, Cu2+, Mn2+) or a trivalent metal cation(e.g., A13+, Gd3+). The organic anion component can be, for example, anorganic acid (e.g., (2-hydroxy-4-methylthio)butanoic acid, or HMTBa), anamino acid (e.g., methionine, glycine, cysteine, phenylalanine), apartially hydrolyzed protein or peptide (e.g., a metal proteinate), apolysaccharide (e.g., a gluconate) or an antibiotic that carries anegative charge (e.g., bacitracin, ciprofloxacin and other quinolones,tetracycline).

Examples of chelated mineral compounds that comprise an organic acidcomponent include, but are not limited to, various MINTREX® organictrace mineral compounds (e.g., MINTREX Zn: Novus International, St.Charles, Mo.). Examples of chelated mineral compounds that comprise anamino acid component include, but are not limited to, various metalmethionine compounds (e.g., ZINPRO compounds, MANPRO compounds: ZinproCorporation, Eden Prairie, Minn.; see also U.S. Pat. No. 4,021,569),zinc-cysteine complexes, zinc-phenylalanine complexes and glycinates.Examples of chelated mineral compounds that comprise a partiallyhydrolyzed protein or peptide component include, but are not limited to,various metal proteinates (e.g., Zinc bacitracin (a cyclic polypeptidemetal ion complex) as well as Zinc Proteinates, Manganese Proteinates,Copper Proteinates: Balchem Corporation, New Hampton, N.Y.). Examples ofchelated mineral compounds that comprise antibiotics include, but arenot limited to, zinc bacitracin, zinc tetracycline and zincciprofloxacin.

In the methods of the invention relating to maintaining calciumhomeostasis, or restoring calcium homeostasis, in an animal, the agentthat adversely affects calcium homeostasis in the animal can be a CaSRmodulator itself, such as, for example, a CaSR agonist (e.g., a chelatedmineral compound). As described herein, certain chelated mineralcompounds can adversely affect calcium homeostasis in an animal (e.g.,by decreasing serum calcium levels when administered to the animal atparticular concentrations). Examples of chelated mineral compounds thatcan adversely effect calcium homeostasis in an animal include, forexample, MINTREX® organic trace mineral compounds, metal methioninecomplexes, metal proteinates, and Zinc bacitracin. Other agents that canadversely effect calcium homeostasis in an animal (e.g., at particularconcentrations) include, for example, inorganic minerals and inorganicmineral sources (e.g., zinc sulfate). Calcium homeostasis and,indirectly protein metabolism and utilization can be adversely affectedby, for example, decreased serum calcium levels, providing for alteredlevels of counter-regulatory hormones (e.g., parathyroid hormone,calcitonin, gastrin and cholecystokinin), changes in the steady state orfluxes in calcium reabsorption from body stores such as bone or avianskin and the excretion of calcium from the body in the case of milkproduction in mammals and egg shell production in avian species.Moreover, there can also be adverse effects on nutrient uptake andutilization that would include calcium uptake from dietary sources aswell as it reabsorption from various tissues including the kidney.

As described herein, for methods of the invention relating tomaintaining and/or restoring calcium homeostasis in an animal, theco-administration and/or prior or subsequent administration of aneffective amount of one or more CaSR modulators can maintain or restorecalcium homeostasis, respectively, in animals whose calcium homeostasishas been adversely effected by prior or co-administration of an agentthat adversely affects calcium homeostasis, such as a chelated mineralcompound. Thus, an effective amount of one or more CaSR modulators isable to counteract (e.g., off-set) the negative effects of the agentthat adversely affects calcium homeostasis when administered to theanimal.

The invention further relates to a method of weaning a young animal,comprising administering one or more CaSR modulator(s) (e.g., CaSRagonist(s)) to the animal in an effective amount to modulate (e.g.,agonize) one or more Calcium-Sensing Receptors (CaSRs) in one or moretissues in the animal (e.g., in the gastrointestinal tract). In anembodiment, the young animal is a young porcine animal. Other suitableanimals include, for example, bovine animals. Preferably the one or moreCaSR agonists employed in the method include a phenylalkylamine (e.g.,fendiline, prenylamine, cinacalcet). In a preferred embodiment, thephenylalkylamine is fendiline.

In addition, the invention provides a method of improving the skin of anavian animal (e.g., a broiler chicken, a turkey), comprisingadministering one or more CaSR modulator(s) (e.g., CaSR antagonist(s))and a source of Vitamin D (e.g., 25-hydroxycholecalciferol) in effectiveamounts to modulate (e.g., antagonize) one or more Calcium-SensingReceptors (CaSRs) in the skin of the animal.

The invention also relates to a method of inhibiting an entericcondition in a non-human animal, comprising administering a one or moreCaSR modulators (e.g., CaSR agonists) to the animal in an effectiveamount to modulate one or more Calcium-Sensing Receptors (CaSRs) in theanimal (e.g., in the gastrointestinal tract). Examples of entericconditions include, but are not limited to, diarrhea, abnormal gutdevelopment and impaired nutrient utilization. In an embodiment, theanimal is a porcine animal or a bovine animal. Other suitable animalsinclude, for example, companion animals, such as dogs, cats, etc.Preferably the one or more CaSR modulators employed in the methodinclude a phenylalkylamine (e.g., fendiline, prenylamine, cinacalcet).In a preferred embodiment, the phenylalkylamine is fendiline. In someembodiments, the one or more CaSR modulators does not includeprenylamine.

Methods of Reducing Foot Lesions in Terrestrial Avian Animals

In another embodiment, the invention relates to a method of preventingfoot (e.g., foot pad) lesions in a terrestrial avian animal. The methodcomprises administering to an avian animal that has ingested, isingesting, or will ingest an agent that adversely affects calciumhomeostasis resulting in foot pad lesions, one or more CaSR modulator(s)in an effective amount to prevent foot pad lesions in the animal. In apreferred embodiment, the terrestrial avian animal is a chicken.

Preferably, the one or more CaSR modulator(s) include at least one CaSRantagonist (e.g., a naturally occurring CaSR antagonist). In oneembodiment, the agent that adversely affects calcium homeostasis is achelated mineral compound. Examples of chelated mineral compoundssuitable for use in the methods include organic acid-metal chelates(e.g., HMTBa compounds with divalent cations), amino acid-metal chelates(e.g., zinc methionine compounds) and metal proteinates (e.g., zincproteinates, copper proteinates, manganese proteinates, zincbacitracin).

In another embodiment, the invention relates to a method of reducingfoot lesions in a chicken by feeding the chicken a food compositioncomprising at least one chelated mineral compound;25-hydroxycholecalciferol (e.g., at a concentration of about 0.05% byweight); and a source of calcium.

Administration of CaSR Modulators

A CaSR modulator can be administered to an animal in a number of ways.Preferably, the CaSR modulator is delivered to the gastrointestinaltract of the animal. For example, a CaSR modulator can be added to anyone of the food sources described herein (e.g., a feed, a liquid, anutritional supplement), or any combination thereof (e.g., in both thefeed and the water that is provided to the animal), such that the CaSRmodulator is delivered to the gastrointestinal tract upon ingestion ofthe food source by the animal, and the CaSR modulators are released upondigestion.

The CaSR modulators can also be administered in suitable oral dosageforms, such as, for example, tablets, capsules (each of which includessustained release or timed release formulations), pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions. Theymay also be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, all using dosageforms well known to those of ordinary skill in the pharmaceutical arts.

For instance, for oral administration of CaSR modulators in the form ofa tablet or capsule, the CaSR modulator(s) can be combined with an oral,non-toxic, pharmaceutically acceptable, inert carrier such as lactose,starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like;for oral administration in liquid form, the CaSR modulator(s) can becombined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

The CaSR modulator(s) can also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles, and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine, orphosphatidylcholines.

For egg-laying animals (e.g., chickens), CaSR modulators can also beadministered in a composition that is injected directly into the eggbefore the animal has hatched. Suitable methods for injecting eggs arewell known in the art. In the case of feathered animals (e.g.,chickens), CaSR modulators can be applied in a composition that issprayed onto the feathers of the animals, such that the CaSR modulatorsare ingested by the animals during preening. Suitable methods forspraying feathers are well known in the art.

Certain types of CaSR modulators (e.g., peptides, small organicmolecules, such as polyamines and phenylalkylamines) can also beincorporated into polymers that are ingested by the animals, such thatindividual CaSR modulators are released from the polymers in thegastrointestinal tract of the animals. For example, a peptide or smallorganic molecule CaSR modulator can serve as the repeating unit of apolymer, wherein the individual CaSR modulators in the polymer arejoined by linkages (e.g., peptide bonds, non-peptide covalent bonds)that occur at regular intervals and are selectively cleaved (e.g.,chemically, enzymatically, hydrolytically), for example, in the stomachand/or intestine of an animal that has ingested the polymer. Thelinkages in the polymers can be susceptible to cleavage under conditionspresent in the target organ (e.g., stomach, intestine), such as acidic(stomach) or alkaline (intestines) pH, or by specific enzymes that arepresent in the target organ (e.g., trypsin). Suitable cleavable linkagesfor use in polymers are known to those of skill in the art.

Polymers of CaSR modulators can be prepared using standard chemicalsynthesis techniques that are well known in the art. For example,standard chemical synthesis techniques could be utilized to createpolymers of repeating units that each possess an appropriate number ofamino groups positioned at appropriate distances such that, after theingested polymer had been acted upon by hydrolytic enzymes that arepresent in the gastrointestinal tract of the animal, the hydrolyzedproducts can stimulate CaSRs in the mucosa of the gastrointestinaltract. An exemplary polymer containing the CaSR modulator, spermine, isillustrated in FIG. 23B and described herein. For example, examinationof the molecular charge distribution of positively charged amino groupson the potent CaSR agonist, spermine, reveals that its 4 amino groupsare positioned at similar intramolecular distances from each other (FIG.23A). Thus, standard chemical synthesis techniques can be utilized tocreate polymers of repeating units that each possess an appropriatenumber of amino groups positioned at appropriate distances such that,after the ingested polymer had been acted upon by hydrolytic enzymesthat are present in the gastrointestinal tract of the animal, thehydrolyzed products (FIG. 23B) can stimulate CaSRs in the mucosa of thegastrointestinal tract.

Polymers of CaSR modulators can be incorporated into coating materialsfor capsules or other enclosures that are ingested by the animals, orcan themselves be encapsulated in a delivery vehicle (e.g., a capsule, atablet, a microparticle, a nanoparticle) and ingested by the animals.Such delivery vehicles preferably release the polymer in the targetorgan (e.g., stomach, intestine) and include both immediate andcontrolled (e.g., sustained, targeted) release formulations. Thus, thelocation of release of the polymer, and therefore the CaSR modulator(s),can be carefully controlled.

For example, the CaSR modulator(s) can be coupled with soluble polymersas targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. The CaSR modulator(s) can also becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacylates, and crosslinked or amphipathicblock copolymers of hydrogels.

Micro and nanoparticles that can encapsulate agents in order to provideprotection and regulate their rate of release are described in U.S. Pat.No. 5,352,461, which relates to the self-assembling particle drugdelivery systems formed from 2,5-diketo-3,6-di(4-succinylaminobutyl)piperazine that disassemble and release the entrapped agent at high pH.Other particles that share the properties of stability at low pH andinstability as pH increases are described in International PatentPublication No. WO 88/01213. Self-assembling pH titratable particulatesystems, based upon the self-assembling properties of bis-amidedicarboxylic acids are described in the work of Bergeron et al., J.Amer. Chem. Soc. 1995, 117, 6658-6665.

International Patent Publication No. WO 96/29991 describes the formationof self-assembling particles that are based upon polyaminoacids, moreparticularly polyleucine-glutamate. These particles which are preparedfrom natural amino acids have the property of controlled particle sizeand are stable over a wide pH range.

Particles for entrapment of agents, and particularly peptides orproteins, can also be formed by polyelectrolyte complexation of variousanionic polymers with cationic polymers. Anionic polymers may includenatural substances such as sodium alginate, carboxymethyl cellulose,guaran, polyglutamic acid and their derivatives, amongst others.Examples of cationic polymers include polylysine and gelatin. Otherpolycations and polyanions are described in detail within EuropeanPatent No. 671169, U.S. Pat. Nos. 4,835,248 and 5,041,291.

The particles containing CaSR modulators described herein (e.g.,microparticles) can, for instance, be absorbed by Peyer's patches in thegut of an animal that has ingested the particles.

In a particular embodiment, the polymer is protected from breakdown inthe stomach of the animal by an enteric coating, which is degraded inthe intestine of the animal, thereby releasing the polymer into theintestines, where the polymer can be cleaved into its CaSR modulatorconstituents. Enteric coatings are those coatings that remainsubstantially intact in the stomach, but dissolve and release thecontents of the dosage form once it reaches the small intestine. Entericcoatings have been used for many years to arrest the release of the drugfrom orally ingestible dosage forms. Depending upon the compositionand/or thickness, the enteric coatings are resistant to stomach acid forrequired periods of time before they begin to disintegrate and permitslow release of the drug in the lower stomach or upper part of the smallintestines. Most enteric coating polymers begin to become soluble at pH5.5 and above, with maximum solubility rates at pHs greater than 6.5.

A large number of enteric coatings have been described and are typicallyprepared with ingredients that have acidic groups such that, at the verylow pH present in the stomach, i.e., pH 1.5 to 2.5, the acidic groupsare not ionized and the coating remains in an undissociated, insolubleform. At higher pH levels, such as in the environment of the intestine,the enteric coating is converted to an ionized form, which can bedissolved to release the proanthocyanidin polymer composition. Otherenteric coatings remain intact until they are degraded by enzymes in thesmall intestine, and others break apart after a defined exposure tomoisture, such that the coatings remain intact until after passage intothe small intestines.

Suitable enteric coatings are described, for example, in U.S. Pat. No.4,311,833 to Namikoshi, et al.; U.S. Pat. No. 4,377,568 to Chopra; U.S.Pat. No. 4,385,078 to Onda, et al.; U.S. Pat. No. 4,457,907 to Porter;U.S. Pat. No. 4,462,839 to McGinley, et al.; U.S. Pat. No. 4,518,433 toMcGinley, et al.; U.S. Pat. No. 4,556,552 to Porter, et al.; U.S. Pat.No. 4,606,909 to Bechgaard, et al.; U.S. Pat. No. 4,615,885 to Nakagame,et al.; and U.S. Pat. No. 4,670,287 to Tsuji.

Preferred enteric coating compositions include alkyl and hydroxyalkylcelluloses and their aliphatic esters, e.g., methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxybutylcellulose, hydroxyethylethylcellulose,hydroxyprophymethylcellulose, hydroxybutylmethylcellulose,hydroxypropylcellulose phthalate, hydroxypropylmethylcellulose phthalateand hydroxypropylmethylcellulose acetate succinate;carboxyalkylcelluloses and their salts, e.g.,carboxymethylethylcellulose; cellulose acetate phthalate;polycarboxymethylene and its salts and derivatives; polyvinylalcohol andits esters, polycarboxymethylene copolymer with sodium formaldehydecarboxylate; acrylic polymers and copolymers, e.g., methacrylicacid-methyl methacrylic acid copolymer and methacrylic acid-methylacrylate copolymer; edible oils such as peanut oil, palm oil, olive oiland hydrogenated vegetable oils; polyvinylpyrrolidone;polyethyleneglycol and its esters, e.g., and natural products such asshellac.

Other preferred enteric coatings include polyvinylacetate esters, e.g.,polyvinyl acetate phthalate; alkyleneglycolether esters of copolymerssuch as partial ethylene glycol monomethylether ester ofethylacrylate-maleic anhydride copolymer or diethyleneglycol monomethylether ester of methylacrylate-maleic anhydride copolymer,N-butylacrylate-maleic anhydride copolymer, isobutylacrylate-maleicanhydride copolymer or ethylacrylate-maleic anhydride copolymer; andpolypeptides resistant to degradation in the gastric environment, e.g.,polyarginine and polylysine. Mixtures of two or more of the abovecompounds may be used as desired.

The enteric coating material may be mixed with various excipientsincluding plasticizers such as triethyl citrate, acetyl triethylcitrate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, dibutyltartrate, dibutyl maleate, dibutyl succinate and diethyl succinate andinert fillers such as chalk or pigments.

The composition and thickness of the enteric coating may be selected todissolve immediately upon contact with the digestive juice of theintestine. Alternatively, the composition and thickness of the entericcoating may be selected to be a time-release coating which dissolvesover a selected period of time, as is well known in the art.

Food Compositions

Any of the CaSR modulators described herein, and combinations thereof,can be added to a food source (e.g., a feed) for an animal. The CaSRmodulator(s) are typically added to the food source in an amount (e.g.,an effective amount) sufficient to produce a significant positive effecton nutrient absorption and/or utilization in an animal that has ingestedthe food source to which the CaSR modulator(s) has been added, providedthat the CaSR modulator(s) are not present in an amount that producessignificant detrimental effects associated with excess CaSR modulationin an animal, including, but not limited to, toxicity, hypercalcemia,hypocalcemia, reduced appetite, disturbances in electrolyte levels,decreased nutrient utilization and/or fluid retention, and tissuedysfunction (e.g., gastrointestinal tissue dysfunction, nervous tissuedysfunction, endocrine tissue dysfunction).

The animals are provided with a food source that contains one or moreCaSR modulator(s) in sufficient amounts to modulate the expression,sensitivity, activity, signalling and/or physiological function of atleast one CaSR in one or more tissues of the animals. The food sourceincludes nutrients utilized by the animal for nourishment and,preferably, is suitable for ingestion by the animal. The food source canbe, for instance, a feed (e.g., a solid feed, a semi-solid feed) foranimal consumption. Standard feeds for various species of animal areknown in the art. The food source can also be a liquid component of ananimals diet (e.g., an aqueous composition, milk, colostrum). Foodsources can also include nutritional supplements, such as, for example,vitamins, cofactors, enzymes, oils, herbs, and medicaments. Suchsupplements can be provided to young animals in, e.g., microparticles,nanopatricles, polymers, coatings, tablets, capsules, pills, cachets,powders, granules, elixirs, tinctures, suspensions, syrups, emulsions,and/or other suitable enclosures for nutrients that are known in theart.

The frequency and amount of feed that an animal is fed can be determinedby those of skill in the art and will vary depending on a number offactors, including the species of the animal, as well as its size, age,weight, health and gender, among other factors.

In one embodiment, the invention relates to a food composition fornon-human terrestrial animal consumption, comprising:

-   -   a.) at least one chelated mineral compound in an amount that        adversely affects calcium homeostasis in the animal; and    -   b.) at least one CaSR modulator in an effective amount to        maintain and/or restore calcium homeostasis (e.g., a desired        calcium homeostasis) in the animal.

Suitable chelated mineral compounds for the food composition include,but are not limited to, compounds comprising HMTBa and a divalent metalcation (e.g., Zn2+, Cu2+, Mn2+), compounds comprising methionine and adivalent metal cation (e.g., Zn2+, Cu2+, Mn2+), a zinc proteinate, acopper proteinate, a manganese proteinate and zinc bacitracin. The atleast one chelated mineral compound can be present in the composition,for example, at a concentration in the range of about 10 parts permillion to about 200 parts per million by weight, preferably, at aconcentration of about 20 parts per million to about 80 parts permillion by weight.

Suitable CaSR modulators for the food composition include, for example,CaSR antagonists (e.g., MC106). The at least one CaSR modulator can bepresent in the composition at a concentration, for example, in the rangeof about 50 parts per million to about 150 parts per million by weight,preferably, at a concentration of about 50 parts per million to about 90parts per million by weight.

In another embodiment, the invention relates to a food composition forchicken consumption, wherein the food composition is useful forpreventing (e.g., inhibiting) lesions on chicken feet, comprising:

-   -   a.) at least one agent that adversely affects calcium        homeostasis in the animal;    -   b.) 25-hydroxycholecalciferol; and    -   c.) a source of calcium.

In one embodiment, the agent is a chelated mineral compound. Examples ofchelated mineral compounds suitable for the chicken food compositions ofthe invention include, but are not limited to, compounds comprising(2-hydroxy-4-methylthio)butanoic acid (HMTBa) and a divalent cation(e.g., Zn2+, Cu2+, Mn2+). The at least one chelated mineral compound canbe present in the composition in an amount that adversely affectscalcium homeostasis in the chicken (e.g., an amount sufficient todecrease serum calcium levels). In some embodiments, at least onechelated mineral compound is present in the composition at aconcentration of about 0.05% to about 0.50% (5 ppm to 50 ppm) by weight.Preferably, the at least one chelated mineral compound is present in thecomposition at a concentration of about 0.10% (10 ppm) by weight.

The 25-hydroxycholecalciferol is present in the composition at a amountthat is effective to reduce foot lesions in the chicken (e.g., an amountthat is effective to maintain or restore a desired calcium homeostasisin the chicken), for example, at a concentration of about 0.02% to about0.10% by weight, preferably, at about 0.05% by weight.

The source of calcium can be an inorganic calcium source or an organiccalcium source. The calcium is present in the composition at a amountthat is effective to reduce foot lesions in the chicken (e.g., an amountthat is effective to maintain or restore a desired calcium homeostasisin the chicken), for example, preferably in a range of about 1.00% toabout 2.00% by weight, more preferably in the range of about 1.40% toabout 1.45% by weight.

For ruminant animals like steers, cows or lambs, chelated mineralcompounds such as zinc, manganese or copper proteinates can be providedas a % of the total trace mineral requirement. In some embodiments,metal proteinates would be added such that their portion would be 15-30%of the total trace mineral requirement that itself would be provided atvarious concentrations depending on the portion that is provided asinorganic mineral constituents.

For monogastric animals such as weaning pigs, chelated mineral compoundssuch as copper, zinc and manganese can be also provided a % of the totaltrace mineral requirement. In some embodiments, metal proteinates wouldbe added at levels of approximately 50-100 parts per million as anappropriate % of the total trace mineral requirement for this species.For other monogastric animals such as dogs and cats similar quantitiesof chelated trace minerals would be provided at various concentrationsdepending on the portion that is provided as inorganic mineralconstituents and the specific life stage of the particular animal.

The invention is further specifically described in the followingexamples.

EXEMPLIFICATION Example 1 Identification, Isolation and Characterizationof a CaSR Protein in the Jejunum of Chickens

Materials and Methods

Tissue Preparation

Gastrointestinal (i.e., duodenum, jejunum and ileum), kidney and braintissues were harvested from 22 day old chickens (n=3), frozen on dry iceand stored at −80° C.

RNA Extraction

Total RNA was purified from the jejunum using STAT-60 (Teltest B,Friendswood, Tex.), and poly A+ mRNA was isolated using theMicro-FastTrack 2.0 Kit (Invitrogen, Carlsbad, Calif.).

Chick Jejunum cDNA Library Construction

cDNA was synthesized by reverse transcribing chick jejunum mRNA,prepared as described above. Selected cDNA fractions were ligated andpackaged as ZAP libraries (ZAP-cDNA synthesis Kit and ZAP-cDNA GigapackIII Gold Cloning Kit, Stratagene, La Jolla, Calif.). DegenerateCaSR-specific DNA primers (dSK-F3: 5′-TGT CKT GGA CGG AGC CCT TYG GRATCG C-3′ (SEQ ID NO:9); dSK-R4: 5′-GGC KGG RAT GAA RGA KAT CCA RAC RATGAA G-3′ (SEQ ID NO:10) were used to selectively amplify a ˜653 bpfragment of a chick CaSR transcript (FIG. 1), which was used as a probefor the isolation of a full length CaSR cDNA from chick jejunum. A totalof 40,000 phage plaques were screened using duplicate filter lifts(Magna Nylon membranes, GE Osmonics) using the ˜653 bp chicken CaSRprobe created using degenerate CaSR primers that was ³²P-labeled withthe RadPrime DNA Labeling System (Invitrogen) to greater than 50%incorporation. Hybridization of the plate-lifts was carried outovernight at 68° C. in 6×SSC, 0.5% SDS, 5×Denhardt's solution, andmembranes were washed under relatively stringent conditions (30 minutesin 2×SSC, 0.1% SDS, 30 minutes in 0.1×SSC, 0.1% SDS, all at 55° C.). Asingle positive plaque was picked, eluted, and plated. These plates werelifted onto nylon membranes and subjected to secondary and tertiaryhybridization screening.

DNA Sequencing

Tertiary picks were excised from their Lambda-Zap phagemids andtransformed into chemically competent DH5a cells. From this point on,the excised clone was in pBluescript SK−. Upon standard miniprep andconfirmation of insert, the clone was midiprepped using the Wizard PlusMidiprep DNA Purification System (Promega) and sequenced usingcommercially available DNA sequencing facilities at the University ofMaine DNA Sequencing Facility.

Genomic Southern Analysis

Genomic DNA from chicken heart tissue was prepared using the WizardGenomic DNA Purification Kit (Promega), and a 10 μg of the DNA wasdigested overnight with EcoRI. The resulting genomic DNA digest wasfractionated using a 0.7% agarose gel which was then stained and thentransferred to a charged nylon membrane (Ambion BrightStar Plus). Theresulting membrane was then hybridized overnight at 68° C. in 6×SSC,0.5% SDS, 5×Denhardt's solution, using a ³²P-labeled probe made from ourfull-length chicken clone (prepared with Invitrogen's RadPrime DNALabeling System to greater than 50% incorporation). The blot was washedas follows: 30 minutes in 2×SSC, 0.1% SDS, 30 minutes in 0.2×SSC, 0.1%SDS, all at 53° C. (relatively stringent for Genomic Southern Blotting).

Results

A full-length cDNA encoding a CaSR from chick jejunum (FIGS. 2A-C) wasisolated from a chick jejunum cDNA library using a 653 bp fragment ofchick CaSR transcript that was selectively amplified using degenerateCaSR specific DNA primers as described above. Analysis of thefull-length cDNA sequence revealed that the CaSR coding region of theclone is 5336 bp in length and contains a full length open reading frameof 5252 bp that is nearly identical to that of the known chickencalcium-sensing receptor (GenBank accession number XM 416491). The DNAsequence shown in FIGS. 2A-C differs from the GenBank sequence by 11bases (nucleotides 329, 1397, 2087, 3818, 4145, 4637, 4740, 5036, 5078,5139, and 5319), and encodes a predicted 1,059 amino acid polypeptide(FIG. 3).

To verify that the CaSR cDNA sequence was contained in the genomic DNAof chickens, Southern blotting analysis was performed as described aboveafter EcoRI digestion of isolated chicken genomic DNA, revealing asingle 5.2 kb EcoRI genomic fragment that hybridized to the CaSR cDNAsequence (FIG. 4).

The putative amino acid sequence of the chicken jejunum CaSR protein wascompared to CaSR proteins cloned from human parathyroid, salmon kidney,cod kidney, shark kidney and lobster genomic DNA by sequence alignment(FIG. 5A-B). All of these CaSR proteins display a high degree ofhomology and possess cysteine amino acids that have been demonstrated tobe important in the overall structural characteristics of the CaSRmolecule. Furthermore, the human and chicken CaSRs share a similarextended amino acid sequence that comprises a cytoplasmic domain incontrast to salmon, cod and lobster, which possess very short ortruncated cytoplasmic domains by comparison. As shown at the bottom ofFIG. 5, the cytoplasmic domain of the shark kidney calcium receptor isintermediate in length. Comparison of selected portions of the aminoacid sequences for human and chicken CaSRs demonstrates that variousantibodies that recognize specific domains on the human CaSR proteinwill likely recognize the corresponding domains on the chicken CaSRsince their amino acid sequences in the region of antibody binding areidentical.

Example 2 Chick Intestinal CaSR Protein Responds to Ca2+ Via DownstreamActivation of the pERK Signal Transduction Pathway

Materials and Methods

Transfection of Chicken CaSR into HEK-293 Cells

HEK-293 cells were plated into 6-well plates so that the cells were 90%confluent at the time of transfection. The linearized and non-linearizedplasmids were ethanol precipitated and quantifiedspectrophotometrically. A ratio of DNA to lipofectamine of 1:3.5 (4 μgof DNA and 14 μl Lipofectamine 2000) was used for the transfectionprocedure. To provide a negative control, only Lipofectamine was addedto some HEK cells. After exposure to lipofectamine, hygromycin B (400μg/ml) was used to select for HEK cells that had been transfected sincethe plasmid containing the putative CaSR sequence also contained ahygromycin resistance element.

At the start of hygromycin selection, flasks were about 40% confluent.All flasks experienced a high percentage of die off cells. After aninterval of 10 days, all the cells in the negative control flasks weredead. By contrast, the flasks with the non-linearized plasmid showedmore colonies compared to the flasks with the linearized plasmid. Linearflask 1 and non-linear flask 1 were trypsinized by addition of 200 μltrypsin to redistribute dense colonies and prevent overcrowding 10 daysafter the start of hygromycin selection. New complete media was addedcontaining 400 μg/ml hygromycin. At day 14, flasks had multiple coloniesthat were well spread out and were beginning to look overcrowded. Eachflask was trypsinized, centrifuged and re-plated in a new flask, stillunder hygromycin selection. At day 17, transfected HEK cells were 60%confluent. On day 18, each flask was split into two flasks. All flaskswere confluent two days later. One flask was harvested forimmunoblotting analysis and the other was distributed into 4 vials perflask and frozen.

Results

To demonstrate that the open reading frame contained in the CaSR cDNAisolated from chick jejunum encodes a functional CaSR protein, thefull-length cDNA clone described in Example 1 and FIGS. 2A-C wastransfected into HEK cells and selected for HEK cells expressing proteinderived from the cDNA using standard hygromycin B selection procedures.A series of cell colonies were identified that survived hygromycin Bselection.

To demonstrate that recombinant CaSR proteins are expressed intransfected HEK cells, homogenates of HEK cells that had been eitherstably transfected with a human CaSR cDNA or HEK cells derived from twodifferent transfections using linearized chicken CaSR cDNA were preparedand subjected to SDS-PAGE and immunoblotting using a specific anti-CaSRantiserum (FIG. 6). As shown by the rightward pointing open arrow inFIG. 6, cells expressing recombinant human CaSR protein displayed abroad immunoreactive band of ˜130 kDa, which has been previouslydescribed. Similarly, homogenates derived from two different transfectedHEK cell pools that were transfected with the chick jejunal CaSR cDNAeach showed two closely spaced immunoreactive bands that co-migrate withthe recombinant CaSR protein (see leftward pointing solid arrow in FIG.6). These data demonstrate that HEK cells transfected with cDNA from thechick jejunum express recombinant chick CaSR protein that is recognizedby specific anti-CaSR antiserum. These data are consistent with DNAsequence information that predicts that the size of the chicken CaSRprotein should be nearly identical to that of human CaSR. Expression ofchick intestinal CaSR in transfected HEK cells was also detected byimmunocytochemistry (FIGS. 7A-D). Chick intestinal CaSR was stronglyexpressed by a few transfected cells in an manner identical to that ofhuman parathyroid CaSR (FIGS. 7B-D).

Exposure of HEK cells transfected with the chick jejunal CaSR cDNA to anincrease in extracellular Ca2+ ions (0.5 mM to 10 mM) produced selectiveactivation of the extracellular regulated kinase (ERK) kinase pathway,as demonstrated using a phospho-ERK-specific antibody that does notrecognize the dephosphorylated form of the ERK kinase (FIG. 8). Toperform this experiment, CaSR-transfected HEK cells or untransfected HEKcells previously bathed in media containing 0.5 mM Ca2+ were thenexposed to an increase in extracellular Ca2+ to 10 mM. Cells were thenprocessed for immunoblotting and membranes probed with antibody specificfor phospho-ERK. Cell fractions from HEK cells transfected with eitherhuman CaSR (rightward pointing open arrow) or two different HEK cellcolonies expressing the chicken CaSR protein (left pointing solid arrow)contained phospho-ERK proteins. By contrast, untransfected HEK cellsexposed to the same increase in extracellular Ca2+ showed noimmunoreactivity with the anti-phospho-ERK antibody (downward pointingopen arrow with asterisk). Taken together, the data shown in FIGS. 6-8demonstrate that the transfected HEK cells express the chick jejunalCaSR protein and that this recombinant CaSR protein is able to respondto increases in extracellular Ca2+ via activation of appropriatedownstream signaling pathways including phosphor-ERK.

Example 3 CaSR mRNA and Protein are Expressed in the Intestine ofChickens During Various Developmental Stages

Materials and Methods

Northern Blot Analysis

Intestinal and kidney tissues were harvested from 3 several week oldchicks and rinsed in RNA later (Ambion) after harvest. Epithelial cellswere scraped from the mucosal surface of each respective intestinalsegment and transferred immediately to Stat 60. Total RNA was isolatedfrom the prep on the same day. To isolate poly A+ RNA, the MicroPoly (A)purist kit (Ambion) was used and the resulting poly-A RNA was quantifiedand precipitated overnight for use in Northern blot analysis thefollowing day. The mRNA was fractionated using the NorthernMaxformaldehyde-based system and a 1% agarose gel that was electrophoresedat 120 volts for 1 hour and 50 minutes. Subsequently, the contents ofthe gel were transferred to a BrightStar membrane for 2.5 hours using adownward transfer assembly and probed using ³²P-labeled CaSR cDNA clonedfrom chick jejunum as described in Example 1.

Immunocytochemistry

Immunocytochemistry was performed on formaldehyde fixed anddeparaffinized sections of intestine from 10 week old chicks usinganti-CaSR specific antiserum. After de-paraffinization, tissues sectionswere incubated in a blocking solution to prevent nonspecific binding andthen incubated for 2 hr or overnight in a solution containing eitheranti-CaSR antiserum or its corresponding pre-immune antiserum obtainedfrom the same animal species. After incubation, the sections were rinsedwith buffer to remove primary antisera and then incubated with secondaryimmune antiserum that will bind to and detect the presence of boundprimary antibody at specific locations within the tissue section. Afterrising the excess secondary antiserum, the section was incubated in acolor development reagent that localizes to the location and presence ofbound antibody. Sections were then mounted and examined using standardmicroscopy techniques.

Results

To evaluate CaSR mRNAs expressed within chick intestine, poly A+ RNAfrom various segments of chick intestine was isolated, fractionated andprobed with ³²P-labeled full length chick intestinal CaSR isolated from22 day old chick jejunum by Northern blotting (FIGS. 9A and B). A CaSRtranscript that appears to co-migrate with the major CaSR mRNAtranscript present in chick kidney was detected in proximal, middle anddistal intestinal segments of chicks that appears to co-migrate with themajor CaSR mRNA transcript present in chick kidney (see solid arrows inFIG. 9A). However, due to the much lower content of CaSR mRNA inintestinal tissue relative to kidney tissue, it appears that the levelof steady state expression of CaSR mRNA in the intestine may be lowerthe intestine than the kidney and perhaps even the parathyroid.

To determine the location of CaSR protein in the proximal intestine ofthe developing chick, CaSR protein was localized to the epithelial cellsof the mucosa and crypt areas by immunocytochemistry (FIG. 10). Thelocation of the CaSR protein within the intestinal epithelia allows forit to be in contact with luminal sources of nutrients and ions that areingested by the chick as well as being expressed by cells in the cryptarea of the intestine where replacement and turnover of epithelial cellsin the mucosa occur. From its location in crypts, CaSR may influence theoverall trophic response of the chick intestine to the presence ofnutrients and ions. Significant CaSR staining was not detected in theunderlying submucosa (see bottom of FIG. 10).

The intestinal segments of chicks of various ages were also analyzed forthe presence of CaSR protein using immunocytochemistry. As shown in FIG.11A, CaSR protein is not very abundant in chicks immediately afterhatching. As noted by the arrows, CaSR protein in the proximal intestineis localized to the epithelial cells (FIG. 11A), while CaSR protein inthe distal intestine is localized to mucus cells (FIG. 11B). It isapparent by the intensity of the CaSR staining that the proximalintestines of older chicks (5 days and 10 weeks) (FIGS. 11C and D)express much more CaSR protein than newly hatched chicks. These datasuggest that a significant increase in CaSR protein expression occursafter the chick has hatched.

Example 4 Addition of CaSR Modulators to the Drinking Water of NewlyHatched Chicks Increases the Expression of CaSR mRNA in their IntestinalTissue

Materials and Methods

mRNA Expression

Newly hatched chicks were divided into two groups. One group wasprovided standard water and feed, while the second group was provideddrinking water that was supplemented with the known CaSR agonistscalcium and tryptophan, and standard feed. After various intervals (days1 and 2), chicks were sacrificed and RNA was prepared from both proximaland distal intestinal segments. Using standard techniques, cDNA wasprepared from the RNA and a 653 bp sequence of CaSR mRNA was selectivelyamplified using PCR and conditions identical to those described inExample 1 above.

Gavage

Male chicks (either newly hatched or 2 weeks of age) were gavaged with asingle application of various doses of the specific CaSR modulator (MC0100), a specific pharmacological modulator of CaSRs, to increase theapparent sensitivity of the CaSR to agonists such as calcium. Anincrease in the apparent sensitivity of the chick CaSR to extracellularcalcium is expected to result in the “resetting” of the normal range ofcalcium within the chick to a lower level of plasma calcium since thefunctioning CaSR are all shifted leftward (more sensitive) on the doseresponse curve of CaSR for calcium.

Results

The addition of a combination of calcium and tryptophan to the drinkingwater of newly hatched chicks produced a significant increase in steadystate CaSR mRNA, as measured by PCR (FIG. 12). As the diet of newlyhatched chicks is primarily carbohydrate-based, the inclusion ofspecific CaSR agonists in the drinking water or diet of newly hatchedchicks may allow for increased expression of CaSR protein and/or mRNA,leading to a variety of possible beneficial effects including growth,feed conversion, immunity, improved nutrient and ion absorption, as wellas intestinal resistance to parasites or bacterial diseases.

To further demonstrate that CaSR proteins in chicks and chickens providecritical nutrient and ion sensing capabilities, as has been demonstratedin humans, newly hatched and 2 week old chicks were subjected to bolustreatments of MC 0100, a specific CaSR modulator that has been wellcharacterized and is known to increase the sensitivity of CaSRs tochanges in extracellular calcium. As shown in FIGS. 13-17, a singlegavage treatment of both 2 week old (FIGS. 13 and 14) and newly hatched(FIGS. 15-17) chicks with various doses of the CaSR modulator producedan expected dose dependent decrease in plasma calcium after variousintervals of time. These data demonstrate that CaSRs in chicks can bemodulated to produce physiological changes in that include alterationsin plasma calcium.

Example 5 CaSRs are Expressed in Epithelial Cells Lining VariousGastrointestinal Tissues of Developing Pigs

Materials and Methods

Stomach and duodenum tissue samples were obtained from a female weanedpiglet (9 hours) of 11.5 lbs. at day 15-16. Immunocytochemistry (ICC)was performed using anti CaSR antibody (4641) at 1:500 dilution asdescribed above.

Results

To determine whether epithelial cells lining the intestine of fetal pigsexpress CaSR protein on their mucosal or serosal surfaces,immunolocalization of CaSR was performed using standard techniques. Asshown in FIGS. 18A and B, epithelial cells lining the piggastrointestinal tract express significant amounts of CaSR protein onthe mucosal surface of the intestine. CaSR protein was also detected inthe epithelial cells lining the antrum (FIG. 19A), stomach (FIG. 19B)and first segment of the intestine (duodenum) (FIG. 19C) of piglets thatwere weaned from sow's milk only 8 hr previously, thereby demonstratingthat CaSR proteins are expressed in stomach and intestinal epithelialcells that are exposed to new dietary substances in piglets.

Example 6 CaSRs Present in the Intestinal Epithelia of Developing Pigscan be Stimulated by Various CaSR Agonists

Background

Under normal conditions, newborn piglets receive most of their nutritionvia the ingestion of milk from their mothers. Published reports (Chenget al., Animal Sci. 82: 95-99 (2006)) of the composition of sowcolostrum and milk demonstrate that they are both rich in CaSR agonistsand possess an ionic composition that would allow for the stimulation ofCaSRs. As shown in FIG. 20A (adapted from Cheng et al.), colostrum andmilk samples obtained during the early stages of milk production containa high content of spermine, a potent CaSR agonist. Although the sperminecontent of milk decreases as days of lactation increase, theconcentration of another potent CaSR agonist, spermidine, increases. Asshown in FIG. 20B, the ionic composition of sow's milk and colostrumalso favor the activation of CaSRs located in the suckling pig'sintestine and stomach via CaSR agonists that are contained in the milk.The low concentration of Na+ ions coupled with the high concentration ofCa2+ ions present in sow's milk has been demonstrated to promote theactivation of CaSRs using recombinant CaSR protein expression studies inHEK cells (Quinn, S M, C-P Yee, R. Diaz, O. Kifor, M. Bai, P. Vassilevand E Brown. Am. J. Physiol. 273:1315-1323, 1997). The elevatedconcentrations of calcium present in sow's milk provide for enhancedstimulation of CaSRs by spermine at concentrations that correspond tothese found in sow milk (FIG. 20C).

Upon the weaning of nursing animals, the nutrient source that willmaintain both the growth of the neonatal animal and its intestinaltissue will be exogenous food sources. Thus, the transition from milk tosolid or semisolid food that occurs upon the weaning of animals isimportant to the future success and agriculture performance of theanimals.

The milk of animals like swine and cows contain high quality nutrientproteins like casein. These nutrient proteins are acted upon by theenzymatic machinery of the gastrointestinal tract of young developinganimals, and peptides derived from the hydrolysis of proteins contactthe mucosal surface of the stomach and intestine. These proteins,peptides and amino acids are absorbed across the intestinal epithelialto be used as nutrients.

Materials and Methods

All methods and equipment utilized to perform experiments on HEK cellshave been reported previously (Gama, L. and Breitwieser, G. E. 1998. Acarboxyl-terminal domain controls the cooperativity for extracellularCa2+ activation of the human calcium sensing receptor. A study withreceptor-green fluorescent protein fusions. J. Biol. Chem. 273,29712-29718; Bai, M., Quinn, S., Trivedi, S., Kifor, 0., Pearce, S. H.,Pollak, M. R., Krapcho, K., Hebert, S. C. and Brown, E. M. 1996.Expression and characterization of inactivating and activating mutationsin the human Ca2+o-sensing receptor. J. Biol. Chem. 271, 19537-19544).Untransfected HEK cells or, alternatively, cells transfected andexpressing CaSR protein were cultured in Dulbecco's Modified EagleMedium containing 10% fetal bovine serum and 1% penicillin-streptomycinin 75 cm² flasks until they reached confluence. Media was removed fromthe flasks and cells were loaded by exposure to Loading Buffer (125 mMNaCl, 4 mM KCl, 1.0 mMCaCl₂, 1.0 mM MgCl₂, 1 mM NaH₂PO₄, 20 mM HEPES, 1gm/liter bovine serum albumin, and 1 gm/l glucose, pH ˜7.4, osmolality˜285) containing 4.1 micromolar FURA2-AM (Molecular Probes Inc. Eugene,Oreg.) for 2 hr. Cells were then scraped from the surface of the flaskusing a standard cell scraper (Costar 3010, Corning Inc.). Cells werethen pelleted from the suspension by low speed centrifugation to removeloading buffer and rinsed twice to remove extracellular FURA2 dye by twosequential suspensions and pelleting steps using low speedcentrifugation. Immediately prior to the start of the experiment, cellswere resuspended in various experimental buffers for analyses asdescribed below. The Standard Experimental Buffer used for many studieswas composed of: 125 mM NaCl, 4 mM KCl, 0.5 mMCaCl₂, 0.5 mM MgCl₂, 20 mMHEPES, 1 g/l glucose, pH ˜7.4, osmolality ˜285. All other buffers werevariations on this basic composition where specific components withinthe Standard Experimental Buffer were varied while the remainingcomponents were held constant.

Cell suspensions consisting of a total volume of 3 ml were analyzed in aPTI fluorimeter (PTI Model 814 Photomultiplier Detection System equippedwith SC-500 Shutter Controller and PTI Driver and Analysis Software)within approximately 20 min after exposure to experimental buffers. Datawas acquired using standard ratio image analysis as described previouslyusing a data acquisition rate of 1.3 sec. for 500 or 1000 secondintervals.

In selected experiments, the detergent Triton X-100 was added in orderto lyse the cells and quantify differences in FURA2 ratio fluorescence.Triton X-100 was added to the cells to obtain a maximal fluorescencesignal from the FURA2 for purposes of quantification.

Results

Casein, a calcium binding protein and constituent of milk, acts as anagonist to recombinant human CaSR protein expressed in HEK cells (FIG.21A), while bovine serum albumin (BSA), a protein that also bindscalcium, has no effect on human CaSR. Similarly, the tripeptideglycine-glycine-arginine (gly-gly-arg) produced a significant activationof recombinant human CaSR protein while exposure of the CaSR to thedipeptide, glycine-phenylalanine (gly-phe), does not activate the CaSRunder identical extracellular ionic conditions (FIG. 21B). Furthermore,exposure of recombinant human CaSR to diaminopropane, a small positivelycharged organic molecule, results in a sharp increase in intracellularcalcium similar to that produced by the exposure of the CaSR toincreases in extracellular calcium (FIG. 22). These data demonstratethat certain milk proteins and their hydrolysis products, as well asspecific peptides from other proteins, can activate CaSR proteinspresent on the mucosal surface of the gastrointestinal tract of nursingneonatal animals.

Example 7 CaSR Modulators Effect the Response of Mammalian and AvianCaSRs to MINTREX® ZN, an Organic Acid Chelated Mineral

Experiments on HEK cells were performed generally as described inExample 6 herein. After an initial addition of 2.5 mM Ca2+ the mammalianCaSR showed a response to the addition of 0.025% Mintrex Zn. Subsequentaddition of Ca2+ to a final concentration of 5 mM produced an additionalresponse (see FIG. 26A). No response was observed upon addition of0.025% Mintrex Zn after the addition of experimental buffer (EB) (seeFIG. 26B). Addition of the CaSR modulator, MC0100, to a finalconcentration of 1 micromolar produced a very small CaSR response andsubsequent addition of 0.025% Mintrex Zn elicited a response as did asubsequent addition of Ca2+ to a final concentration of 5 mM. The CaSRresponded to 0.025% Mintrex Zn only after the addition of 1 micromolarMC0100 (see FIG. 26D), as no response to Mintrex Zn stimulation wasobserved in the absence of previous addition of MC0100 (FIG. 26B). Inaddition, the magnitude of the CaSR response to 5 mM Ca2+ was diminishedby previous addition of Mintrex Zn. This reduction in response ispartially restored by previous addition of MC0100 (FIG. 26D).

Similar results were obtained for the avian CaSR. The CaSR modulatorMC0100 (calcimimetic) potentiated the response of the avian CaSR to thechelated mineral Mintrex Zn. Two additions of experimental buffer (EB)to cells suspended in buffer containing 1.5 mM Ca2+ did not produce aresponse by the avian CaSR. However, after addition of 7.5 mM Ca2+ tothe same cell suspension, there was a response by the avian CaSR asindicated by the large upward deflection of the curve (FIG. 27). Bycontrast, after an identical addition of EB, addition of 0.25% MintrexZn produced a response from the CaSR followed by a diminished responseto the addition of Ca2+ to a final concentration of 7.5 mM. A thirdexperimental run from the same collection of cells yielded a responseupon addition of MC0100 to a final concentration of 1 micromolarfollowed by a significantly larger response of the avian CaSR uponaddition of 0.25% Mintrex Zn. However, the subsequent CaSR response toCa2+ addition to 7.5 mM was reduced. All 3 tracings are derived from asingle pool of cells.

The CaSR antagonist, MC106, a calcilytic, reduced or eliminated the CaSRresponse to either Ca2+ or the chelated mineral, Mintrex Zn. After aninitial addition of dimethylsulfoxide (DMSO) addition of Ca2+ to a finalconcentration of 2.5 mM elicited a response by the CaSR that is alsopresent upon subsequent addition of Ca2+ to a final concentration of 5mM (FIG. 28A). By contrast, addition of 1 micromolar MC106 suspended inthe same concentration of DMSO did not itself produce a CaSR responsebut eliminated the CaSR response to 2.5 mM Ca2+ and reduced thesubsequent response to 5 mM Ca2+ (FIG. 28A).

The addition of DMSO elicited no CaSR response itself while subsequentaddition of 0.25% Mintrex Zn and 5.5 mM Ca2+ produced responses by themammalian CaSR. By contrast, initial addition of 1 micromolar MC106 inthe same concentration of DMSO as before did not elicit a CaSR responsebut also reduced or eliminated responses by the CaSR to either 0.25%Mintrex ZN or 5.5 mM Ca2+ (FIG. 28B). The final addition of thedetergent Triton X-100 served as an internal control in that it lysesthe HEK cells and liberates all FURA2 dye for calibration purposes.

Example 8 CaSR Modulators Effect the Response of Mammalian and AvianCaSRs to ZnPro, an Amino Acid Chelated Mineral

The ability of the CaSR modulator MC0100 to potentiate the response ofavian and mammalian CaSRs to the chelated mineral amino acid complex,ZnPro, was tested. For avian CaSR experiments, three individual aliquotsfrom a single pool of HEK cells that stably express the recombinantavian CaSR protein were used for ratio imaging fluorimetry assays ofCaSR activation generally as described in Example 6 herein. Twoadditions of experimental buffer (EB) to cells suspended in buffercontaining 0.5 mM Ca2+ did not produce a response by the avian CaSR(FIG. 29A). By contrast, after addition of the CaSR modulator, MC0100followed by addition of either 0.02% ZinPro or 0.2% ZnPro producedresponses from avian CaSRs. Subsequent addition of Ca++ to a finalconcentration of 5 mM also produced a response. In all aliquots, thedetergent Triton X-100 was added as the last addition to lyse the cells(FIG. 29A).

For mammalian CaSR experiments, two individual aliquots from a singlepool of HEK cells that stably express the recombinant mammalian CaSRprotein were used for ratio imaging fluorimetry assays of CaSRactivation generally as described in Example 6 herein. Addition of Ca++to a final concentration of 2.5 mM produced a CaSR response that wasalso followed by responses after subsequent additions of 0.025% Znproteinate followed by Ca++ to a final concentration of 7.5 mM (FIG.29B). By contrast, addition of 1 micromolar MC0100, a CaSR agonist, didnot itself produce a large response but instead increased the responseof the CaSR to the same dose of 0.025% Zn Proteinate. There was also aresponse to a subsequent dose of Ca++ to a final concentration of 7.5 mM(FIG. 29B). In both tracings, the detergent Triton X-100 was added as afinal step to lyse the cells.

Example 9 CaSR Modulators Effect the Response of Mammalian and AvianCaSRs to Zinc Proteinate

Experiments on HEK cells were performed generally as described inExample 6 herein. The effects of the CaSR Modulators MC0100 and MC106were compared on avian vs. mammalian CaSRs stimulated by either zincproteinate or the zinc organic acid complex, Mintrex Zn. Multiplealiquots of HEK cells stably expressing either the avian CaSR (FIGS. 30Aand C) or mammalian CaSR (FIGS. 30B and D) were used to perform ratioimaging fluorimetry to determine the effects of CaSR modulators MC0100or MC106 on CaSR stimulation by chelated minerals. Prior addition ofCa++ to a final concentration of 2.5 mM or 1 micromolar produced asimilar response of the avian CaSR as did experimental buffer (EB) alonewhen 0.25% Zinc Proteinate was then added to the cells (FIG. 30A).Subsequent addition of additional Ca++ to a final concentration of 7.5mM to any of the 3 aliquots (EB alone; MC0100 or 2.5 mM Ca++) producedlittle or no CaSR response. By contrast, prior addition of 1 micromolarMC106 reduced the avian CaSR's response to Zn Proteinate (FIG. 30A). Asimilar analysis of the effects of prior addition of Ca++, MC0100 orMC106 on the response of the mammalian CaSR to Zinc Proteinate wasperformed. A similar pattern of inhibition by MC106 on the CaSR responseto Zinc Proteinate was observed (FIG. 30B). Similar results wereobtained for the avian and mammalian CaSRs using the zinc organic acidchelate, Mintrex Zn (FIGS. 30C, D).

Example 10 Zn Proteinate, a Divalent Metal Ion-Proteinate Complex,Activates Both the Avian and Mammalian CaSRs in a Dose ResponseRelationship in a Manner Similar to Mintrex Zn

From a single pool of HEK cells stably expressing the avian CaSR,aliquots of cells were removed and analyzed using ratio imagingfluorimetry generally as described in Example 6 herein. After a standardaddition of Ca++ to a final concentration of 2.5 mM, either 0.0025%,0.025% or 0.25% Zinc Proteinate was added followed by a second additionof Ca++ to a final concentration of 7.5 mM. Subsequently, an aliquot ofthe detergent Triton X-100 was added to lyse the cells. 0.0025% elicitedlittle or no CaSR response while 0.025% and 0.25% produced increasingresponses from the avian CaSR (FIG. 31A). The magnitude of thesubsequent CaSR response to Ca++ was reduced via increasing ZnProteinate stimulation. The mammalian CaSR also displayed a similar doseresponse pattern as the avian CaSR (FIG. 31B). Similar results wereobtained for the avian and mammalian CaSRs using the zinc organic acidchelate, Mintrex Zn (FIGS. 31C, D).

Example 11 The CaSR Modulator L-Phenylalanine (L-Phe) Reverses theInhibitory Effect of Chelated Minerals on Response to a Ca++ Stimulus inMammalian and Avian CaSRs

Experiments on HEK cells were performed generally as described inExample 6 herein. Addition of 3 mM L-Phenylalanine (L-Phe) reversedMintrex-Zn's inhibition of the avian CaSR's response to a Ca++ stimulus.Prior addition of 0.25% Mintrex Zn to the recombinant chicken CaSRexpressed in HEK cells produced an initial receptor response, but nosubsequent CaSR response was obtained after the addition of Ca++ to afinal concentration of 7.5 mM (FIG. 32). The detergent Triton X-100 wasadded last to lyse the cells. By contrast, identical fluorimetryanalysis of a separate aliquot from the same pool of cells revealed thataddition of 3 mM L-Phe after CaSR stimulation with 0.25% Mintrex Znproduced a response to addition of Ca++ to 7.5 mM (FIG. 32). Thus, prioraddition of L-Phe reversed or rescued the avian CaSR from its inhibitionby the chelated mineral Mintrex Zn.

In a separate experiment, addition of 3 mM L-Phenylalanine (L-Phe)reversed Mintrex-Zn's inhibition of the mammalian CaSR's response to aCa++ stimulus at extracellular calcium concentrations that correspond tomammalian serum (1.5 mM Ca++). Addition of experimental buffer alone(EB) produced no response by the recombinant mammalian CaSR expressed inHEK cells (FIG. 33). However, addition of 0.025% Mintrex Zn produced aninitial receptor response, but no subsequent CaSR response was obtainedafter the addition of Ca++ to a final concentration of 5 mM. Thedetergent Triton X-100 was added last to lyse the cells. By contrast,identical fluorimetry analysis of a separate aliquot from the same poolof cells revealed that addition of 3 mM L-Phe after CaSR stimulationwith 0.025% Mintrex Zn produced a response to addition of Ca++ to 5 mM(FIG. 33). Thus, in a manner similar to that shown for the avianreceptor, prior addition of L-Phe reversed or rescued the mammalian CaSRfrom its inhibition by the chelated mineral Mintrex Zn.

Example 12 Mammalian and Avian CaSR Proteins are Able to Respond toMetal Proteinates Via Activation of Downstream Signaling Pathways LikeCaSR Dependent ERK 1/2 Phosphorylation

Materials and Methods

HEK cells transfected with the chicken CaSR were cultured under standardconditions (DMEM with 10% FCS). Confluent flasks were serum-starvedovernight in DMEM supplemented with 0.2% BSA and pre-incubated for 60min at 37° C. in the following medium: 125 mM NaCl, 4 mM KCl, 20 mMHepes, 0.1% glucose, 0.8 mM NaH2PO4, 1 mM MgCl₂, 0.1% BSA, 0.5 mM CaCl₂,pH 7.45. The cells were then incubated in the same medium without BSA inthe presence of different mineral chelates at different concentrations(see below) for 15 min at 37° C. At the end of the incubation treatmentmedia was removed and cells were washed with PBS and than lysed in 250microliter of M-PER lysis buffer (Pierce Chemical Company, Rockford,Ill.) containing 10 ul/ml of protease inhibitors (Pierce ChemicalCompany, Rockford, Ill.). Cell lysates were centrifuged for 10 min at 4°C. at 14,000×g. The protein content in the supernatants was analyzedusing the BCA Protein Assay system from Pierce Chemical Company,Rockford, Ill. Equal amounts of protein (20 ug) were combined with 4×loading buffer and 100 uM DTT, heated to 95° C., separated by SDS gelelectrophoresis and electrotransferred to PVD membranes. The membraneswere incubated in 1% BSA in TBS-T (TRIS-buffered saline containing Tween20 at 0.1%) overnight. After 4 washes in TBS-T the membranes wereincubated with primary antibody (anti phosphoERK at 1:400 dilution; thisantibody recognizes the phosphorylated form of ERK 1/2) for 1.5 hours atroom temperatures, washed 4 times and incubated with secondary antibody(anti mouse at 1:50,000 dilution). After 4 washes in TBS-T the membraneswere developed using the ECL Western Blotting system.

The experiments described in FIGS. 34 A and B used the following metalchelate treatments: ZnProteinate, CuProteinate and MnProteinate (BalchemCorporation, New Hampton, N.Y.) at the following concentrations: 0.025%and 2.5%, or at a concentration of 0.025% with 1 micromolar MC 0100 (acalcimimetic); or various concentrations of bacitracin Zn.

Results

All 3 chelated mineral complexes displayed a dose dependent response.The Zn Proteinate (FIG. 34A) and Mn Proteinate (FIG. 34B) resulted in anincrease in ERK1/2 phosphorylation with increasing concentration. Bycontrast, Cu Proteinate displayed the highest ERK1/2 phosphorylationafter exposure of HEK cells to the lowest concentration of 0.025% (FIG.34A). The calcimimetic MC 0100 strongly enhanced the ERK1/2phosphorylation response to 0.025% Mn Proteinate and to a lesser degreeto Zn Proteinate. Notably, Zn Proteinate at 0.025% already showed ahigher phosphorylation response when compared to the response to 0.025%Mn Proteinate. By contrast, the addition of MC 0100 in combination with0.025% Cu Proteinate results in a net reduction of the ERK1/2phosphorylation response when compared to other metal chelate complexestested.

Exposure of HEK cells stably expressing the chicken CaSR to bacitracinZn resulted in an increase in ERK1/2 phosphorylation as compared to Ca++with MC0100 (FIG. 35).

Example 13 The Phenylalkylamine Compounds Fendiline and PrenylamineStimulate Avian and Mammalian CaSRs in a Manner Similar to MC0100

Fluorimetry analysis was performed on HEK cells expressing therecombinant chicken CaSR protein generally as described in Example 6herein.

In a first experiment, after addition of 2.5 mM Ca++, a series ofstepwise additions of the CaSR agonist, MC0100, caused increasedresponses of the mammalian CaSR (FIG. 36). In a similar manner,additions of fendiline to a second aliquot of the same CaSR-HEK cellsproduced similar responses in the avian CaSR (FIG. 37). As a control, nosuch responses were observed when additions of the vehicle DMSO only wasadded followed by a single addition of Ca++ to a final concentration of5 mM. The addition of the detergent Triton X-100 was added last to eachcuvette to lyse the cells at the end of each analysis run.

In another experiment, 3 separate aliquots obtained from a single poolof CaSR-HEK cells were treated with either 1 micromolar, 3 micromolar or5 micromolar prenylamine after addition of Ca++ to a final concentrationof 2.5 mM. After the addition of prenylamine, each aliquot of cellsreceived a subsequent addition of Ca++ to a final concentration of 7.5mM. A CaSR response was observed for both the mammalian and avian CaSRs(FIGS. 38 and 39). As a control, no such response were observed whenaddition of the vehicle DSMO was added. The addition of the detergentTriton X-100 was added last to each cuvette to lyse the cells at the endof each analysis run.

Example 14 CaSR Proteins Localize to Stratum Corneum, and Epidermal andDermal Tissues, in the Skin of Broiler Chicken Feet

Materials and Methods

Standard immunocytochemistry was performed on permeabilized fixed tissuesections of broiler chicken skin from the foot pad region of feet usingeither specific anti-CaSR antiserum or pre-immune anti-CaSR antiserum.

Results

The presence of CaSR was detected in stratum corneum, epidermis anddermal skin regions (FIG. 40A) and within the dermis (FIG. 40C) of theskin of broiler chicken feet.

Example 15 42-Day Broiler Study

This MariCal broiler trial was conducted to investigate the benefits ofchelated minerals in commercial broiler production.

Principal Objective: The principal objective of this 42 day floor penstudy was to determine if the CaSR modulators MC 0100 or MC 106 producealterations in broilers that were subjected to zinc depletion and thenreceived oral zinc in the form of Mintrex Zn. The primary endpoints ofthe study were the measurement of growth, feed utilization and footpadscores in male broiler chickens raised under conditions resembling thoseused for commercial poultry production (floor pens with used litter).

Study Design: The study was divided into 3 phases for variousexperimental and control groups. All groups were sequentially fedidentical base feeds that included starter (until Day 25), grower (Days26-30) and finisher (Days 31-42) formulations that differed only intheir zinc content or source. For the 4 experimental groups (B-E), these3 phases included a 10 day interval of zinc depletion followed by a zincrepletion phase to Day 25 followed by rearing to Day 42. The 5th controlgroup (Group A) was not subjected to an initial interval of zincdepletion and received a standard commercial diet until Day 25. The MC100 and MC 106 compounds were administered in feed in combination withMintrex Zn and compared to experimental treatment groups receivingeither Mintrex alone (Mintrex ZN) or a low inorganic zinc-content diet(Zn Low). These groups were compared to the corresponding control groupthat was fed a standard inorganic-zinc content diet until day 25. Afterevaluation and analysis on Day 25, all study groups were continuedthrough Day 42 without the addition of the MC compounds.

Protocol details for Groups A-E are outlined in Tables I-IV. Group A wasnot subjected to zinc depletion and was fed standard feed containingsupplemental zinc (125 ppm zinc sulfate). After zinc depletion, each ofthe Groups B-E was fed different zinc replete diet. The diet of Group Bwas supplemented with 40 ppm zinc as zinc sulfate. By contrast, the dietof Group C was supplemented with Mintrex Zn alone (20 ppm supplementalZn). The diet of Group D contained the same Mintrex Zn as in Group Cplus MC 0100 (10 mg/kg BW or 70 mg/kg feed) while the diet fed to GroupE contained Mintrex Zn plus MC 0106 (10 mg/kg BW or 70 mg/kg feed).After analysis on Day 25, the feeds of Groups C-E contained Mintrex Znonly without MC 100 or MC106.

TABLE I Designation of Treatment Groups and sampling schedule (*Growerand Finisher zinc content see below) Treatment Lethal Group Bleed DayTreatment N A D 25, D 43 Regular feed (zinc-supplemented throughout 8pens study at 100 ppm Zn as zinc sulfate*) B D 25, D 43 Low-content zincfeed (zinc-supplemented at 40 8 pens ppm Zn as zinc sulfate starting onDay 10) C D 24, D 42 Mintrex Zn (zinc-supplemented at 20 ppm Zn 8 pensas Mintrex Zn starting on Day 10) D D 24, D 42 MC 0100 (70 ppm infeed) + Mintrex Zn (zinc- 8 pens supplemented at 20 ppm Zn as Mintrex Znstarting on Day 10) E D 24, D 42 MC 0106 (70 ppm in feed) + Mintrex Zn(zinc- 8 pens supplemented at 20 ppm Zn as Mintrex Zn starting on Day10)

Table II shows the feeding schedule and targeted concentrations of zincdesigned for each of the treatment groups.

Table III shows the results of confirmatory QC zinc content analyses foreach of the feeds conducted after completion of the trial by an outsidecontract analysis laboratory. Note that this analysis revealed the zinccontent of all feeds were appropriate with the exception of Group Awhere a misformulation in both the grower and finisher diets provided nosupplemental zinc as required by the protocol. Thus, data from the GroupA control broilers is only valid for this comparison study from Day 0-25and not thereafter.

TABLE II Feeding schedule and targeted concentration of added zinc infeed for each Treatment Group A B C D E inorganic inorganic MintrexMintrex Zn + Mintrex Zn + Zn Zn Zn MC 0100 MC 0106 Day 0-9 100ppm * * * * Starter Feed Day 10- 100 ppm 40 ppm 20 ppm 20 ppm 20 ppm24/25 (1) (2) Starter Feed Day 25-30 100 ppm 40 ppm 20 ppm 20 ppm 20 ppmGrower Feed Day 31-42 100 ppm 40 ppm 20 ppm 20 ppm 20 ppm FinisherFeed * No supplemental zinc added (1) MC 0100 added to feed at 70 ppm(2) MC 0106 added to feed at 70 ppm

TABLE III Feeding schedule and actual concentration of zinc in feed foreach Treatment Group A B C D E inorganic inorganic Mintrex Mintrex Zn +Mintrex Zn + Zn Zn Zn MC 0100 MC 0106 Day 0-9 128.5 ppm  36.1 ppm*  36.1ppm*  36.1 ppm*  36.1 ppm* Starter Feed Day 10- 128.5 ppm 72.6 ppm 64.2ppm 58.8 ppm 60.4 ppm 24/25 (1) (2) Starter Feed Day 25-30  39.7 ppm*78.8 ppm 57.3 ppm 57.3 ppm 57.3 ppm Grower Feed Day 31-42  32.9 ppm*71.9 ppm 54.2 ppm 54.2 ppm 54.2 ppm Finisher Feed *No supplemental zincadded (1) MC 0100 added to feed at 70 ppm (2) MC 0106 added to feed at70 ppm

Table IV summarizes the diet composition for Starter, Grower andFinisher feed used in this trial. For Trace Minerals a special Zn-freemix provided by NOVUS was used in all feed types and supplemented withzinc at specific target concentrations (see Tables II&III).

TABLE IV Diet composition of Trial Feeds (g/kg) Starter Grower FinisherFEED TYPE g/kg g/kg g/kg Corn, Yellow #2 598 Corn AS 101 14 645 701Soymeal 48% 308 Soybean Meal A 265 207 Pork Meat&Bone 50.00 PorkMeat&Bone 50.00 56.93 Fat. Animal Stb 23.62 Animal Fat 340 23.18 22.82Deflour Phosph 5.01 Salt 4.31 4.68 Limestone 4.59 Limestone 4.10 3.14Salt 96+% 4.08 Deflour Phosph 3.50 0.00 Methionine DL 2.20 DL-Methionine2.32 1.75 Vitamins.Broiler¹⁾ 1.00 Poultry Vitamin 1.00 1.00 TraceMinerals 1.00 Poultry Trace 1.00 1.00 Min choline Cl-60 0.94 CholineChloride 0.87 0.57 L-Lysine HCl 0.91 L-Lysine 0.59 0.17 Calcium (%)0.890 0.82 0.72 Avail Phosphorus 0.420 0.39 0.35 (%) ¹⁾Vitamin premixprovides (mg kg⁻¹ diet): thiamin, 1.9; riboflavin, 7.7; pantothenicacid, 12.1; niacin, 27.6; pyridoxine, 3.1; folacin, 1.00; biotin, 0.088;vitamin B₁₂, 0.014; vitamin K, 1.93; vitamin A, 10,472 IU; vitamin D,3031 IU.

Animals

One day old broiler chicks (Cobb 500) were randomly allocated (8 pensper treatment in a block design with 5 pens per block and 27 birds/pen).Following their pen placement, all chicks from Groups B-E were zincdepleted by feeding a low zinc-content diet for 9 Days. By contrast,Group A received standard starter feed with supplemental zinc (see TableIII) until Day 25. On Day 10 the bird count was adjusted to 24 in eachpen and feed containing the respective experimental treatments (seeTables I-III) was administered until Day 25. After sampling on Days 24and 25, the bird count was adjusted to 14 in each pen (feed treatmentssee Table II&III). The birds were allowed free access to both feed andwater ad libitum. Feed consumption was quantified on Days 10, 24, 30 and42. Individual bird weights were determined on Days 10, 24 and 42 andpen weights were taken on Days 25 and 30.

Sampling and Processing

On Day 24, eight birds were selected randomly from each pen fromTreatment Groups C, D, and E and whole blood (−3 ml) was obtained byheart puncture. The blood was allowed to clot and serum was separated bycentrifugation (IEC HN-SII centrifuge, 10 min at 2000 rpm at RT). Serumparameters (ionized calcium, sodium, chloride and potassium) weredetermined the same day using the ABL 70 Radiometer (Radiometer,Copenhagen). The remaining serum was frozen and stored at −20° C. untilanalysis of additional parameters (see below). At the time of bloodsampling, the footpads of each birds were scored for severity of lesionsby a single experienced analyst (scores 0-3). Tissue samples (midjejunum and ileo-cecal junction) were removed from 16 randomly selectedbirds from each group within 5-15 min after heart puncture.

On Day 25 eight birds were selected randomly from each pen in TreatmentsA and B and processed as described above.

At the end of the trial (Days 42 and 43) the same number of birds weresampled as described above.

Serum Analysis

Total protein (Pierce 660 nm Protein Assay) and total calcium,magnesium, phosphorus and glucose (Stanbio Laboratories) were determined(triplicates/sample) using commercial kits and a plate reader (Versamax,Molecular Devices).

Statistical Methods

Data were analyzed using the general linear models procedure in SYSTAT10 followed by Tukey HSD Multiple Comparisons.

Results

Male Cobb strain broiler chickens were reared in standard minipens andfed standard feed. As part of the feed additives, either MC0100 (10mg/kg) or Mintrex Zn (250 mg/kg or MC 106 (10 mg/kg) were included inindividual groups of birds. Blood samples were obtained by heartpuncture on Day 14 of the study and the Serum calcium concentrationsdetermined using a radiometer blood gas instrument (Radiometer,Copenhagen). As compared to Control values, significant reductions inthe mean serum calcium concentration was observed for the two groups ofbirds receiving either MC0100 or Mintrex Zn as a feed additive (FIG.41). No differences in serum pH, Na+, K+ or Cl− were observed for any ofthe groups tested.

A comparison of performance parameters for broiler chickens reared underdirty litter conditions and subjected to zinc depletion for a 10 dayinterval prior to grow out for a total of 42 days was performed. A totalof 1080 male Cobb strain birds were divided into 5 different treatmentgroups (FIG. 42A). These 5 groups were subjected to identical rearingconditions that included being reared using dirty litter conditions tosimulate standard large scale chicken rearing facilities. The 5 groupsincluded a Control group (A) that received standard feeds which included100 ppm inorganic zinc throughout the 42 day grow out. By contrast, theremaining 4 groups were subjected to a 10 day interval of zinc depletionwhereby the zinc content of their food was reduced to minimal levels.Following this interval of zinc depletion, the birds received a 14 dayinterval of zinc repletion using different sources of dietary zinc.These included either inorganic zinc (Group B-40 ppm inorganic zinc),Mintrex Zn (Group C—20 ppm Zinc), Mintrex Zn+MC0100 (Group D—20 ppmZinc; 70 ppm MC0100) or Mintrex Zn+MC106 (Group E—20 ppm zinc; 70 ppmMC106). After a total of 24 days of grow out, Groups C-E all receivedMintrex Zn only (20 ppm Zinc) until their harvest analysis at day 42 ofage. Performance data for Groups A-E is shown FIG. 42B and includes meanvalues for both average weight and adjusted feed conversion ratio (FCR).While there were no significant differences in the means between the 5groups, the group that achieved the largest mean body weight and lowestFCR was Group E, which received the combination of Mintrex Zn and MC106.

A comparison of the frequency and severity of foot pad lesions presentin broiler chickens after 24/25 days of rearing under dirty litterconditions and, in selected cases, subjected to a 10 day interval ofzinc depletion. A total of 1080 Cobb strain birds were divided into 5different treatment groups as described above. On days 23 and 24 of growout, the foot pads of all groups were examined and the frequency andseverity of foot pad lesions were determined by an experienced animalveterinarian. The severity of the foot pad lesions present was graded ona scale from 0 (no lesions present), 1 (mild lesions present), 2(moderate lesions present) and 3 (severe lesions present). As comparedto the other 4 experimental groups, the group receiving the combinationof Mintrex Zn+MC106 displayed a significant reduction in the severityand frequency of foot pad lesions as compared to all other groups (FIG.43A). In addition, there was a significant reduction in the mean footpad severity score of the group receiving Mintrex Zn+MC 106 as comparedto all other groups (FIG. 43B).

A comparison of the frequency and severity of foot pad lesions presentin broiler chickens after 42/43 days of rearing under dirty litterconditions and, in selected cases, subjected to a 10 day interval ofzinc depletion, was determined. The group receiving the combination ofMintrex Zn+MC 106 displayed a significant reduction in the severity andfrequency of foot pad lesions as compared to all other groups (FIG.44A). There was a significant reduction in the mean foot pad severityscore of the group receiving Mintrex Zn+MC106 as compared to all othergroups (FIG. 44B).

A comparison of the ionized and total serum calcium of broiler chickenson Day 24/25 of a 42 Day Grow Out. Birds receiving the combination ofMintrex Zn+MC0100 displayed a ionized calcium concentration that wassignificantly lower than all other test groups (FIG. 45A). Mean valuesfor the total serum calcium concentration for each of the 5 test groupswere obtained from the same samples used for determination of ionizedcalcium shown in FIG. 45A. The birds receiving the combination ofMintrex Zn+MC106 displayed the highest total calcium concentrations(FIG. 45B).

The relevant teachings of all patents, published applications andreferences cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example and preferred embodiments thereof, it will beunderstood by those skilled in the art that various changes may be madetherein without departing from the scope of the invention encompassed bythe appended claims and equivalents thereof.

What is claimed is:
 1. A method of inhibiting foot pad lesions in anon-human terrestrial animal, comprising administering to the animal aneffective amount one or more Calcium Sensing Receptor (CaSR)modulator(s), thereby inhibiting foot pad lesions in the animal.
 2. Themethod of claim 1, wherein the one or more CaSR modulator(s) is a CaSRantagonist.
 3. The method of claim 2, wherein the CaSR antagonist is aphenylalkylamine.
 4. The method of claim 3, wherein the phenylalkylamineis MC106.
 5. The method of claim 1, wherein the one or more CaSRmodulator(s) is a naturally occurring compound.
 6. The method of claim1, wherein the animal has ingested an agent that adversely affectscalcium homeostasis.
 7. The method of claim 6, wherein the agent is achelated mineral compound.
 8. The method of claim 7, wherein thechelated mineral compound comprises at least one divalent cationselected from the group consisting of Zn2+, Cu2+and Mn2+.
 9. The methodof claim 7, wherein the chelated mineral compound comprises(2-hydroxy-4-methylthio)butanoic acid (HMTBa) and Zn2+.
 10. The methodof claim 9, wherein the chelated mineral compound is MINTREX®Zn.
 11. Themethod of claim 7, wherein the chelated mineral compound comprisesmethionine or glycine and Zn2+.
 12. The method of claim 7, wherein thechelated mineral compound is selected from the group consisting of azinc proteinate, a copper proteinate, a manganese proteinate and zincbacitracin, or a combination thereof.
 13. The method of claim 1, whereinthe non-human terrestrial animal is a mammal.
 14. The method of claim 1,wherein the non-human terrestrial animal is an avian animal.
 15. Themethod of claim 14, wherein the avian animal is selected from the groupconsisting of a chicken, a turkey, a duck, a goose, a pheasant, a grouseand an ostrich.
 16. The method of claim 14, wherein the avian animal isa duck.
 17. A method of inhibiting a foot pad lesion in anon-terrestrial avian animal, comprising administering a foodcomposition to the animal, wherein the food composition comprises: a.)at least one chelated mineral compound in an amount that adverselyaffects calcium homeostasis in the animal; b.) 25-hydroxycholecalciferolat a concentration of at least about 0.05% by weight; and c.) a sourceof calcium.
 18. The method of claim 17, wherein the at least onechelated mineral compound comprises (2-hydroxy-4-methylthio)butanoicacid (HMTBa) and a divalent cation selected from the group consisting ofZn2+, Cu2+and Mn2+.
 19. The method of claim 17, wherein the at least onechelated mineral compound is present in the composition at aconcentration of at least about 5 to about 50 parts per million.
 20. Themethod of claim 17, wherein the source of calcium is present in thecomposition at a concentration of about 1.40% to about 1.45% by weight.